Tetrameric cytokines with improved biological activity

ABSTRACT

The present invention concerns methods and compositions for forming cytokine-antibody complexes using dock-and-lock technology. In preferred embodiments, the cytokine-MAb DNL complex comprises an IgG antibody attached to two AD (anchor domain) moieties and four cytokines, each attached to a DDD (docking and dimerization domain) moiety. The DDD moieties form dimers that bind to the AD moieties, resulting in a 2:1 ratio of DDD to AD. The cytokine-MAb complex exhibits improved pharmacokinetics, with a significantly longer serum half-life than either naked cytokine or PEGylated cytokine. The cytokine-MAb complex also exhibits significantly improved in vitro and in vivo efficacy compared to cytokine alone, antibody alone, unconjugated cytokine plus antibody or cytokine-MAb DNL complexes incorporating an irrelevant antibody. In more preferred embodiment the cytokine is G-CSF, erythropoietin or INF-α2b.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/752,649, filed Apr. 1, 2010, which was a continuation-in-part of U.S.patent application Ser. No. 12/418,877, filed Apr. 6, 2009, whichclaimed the benefit under 35 U.S.C. 119(e) of U.S. Provisional PatentApplication Ser. Nos. 61/043,932, filed Apr. 10, 2008, 61/104,916, filedOct. 13, 2008 and 61/119,542, filed Dec. 3, 2008, and which was acontinuation-in-part of U.S. patent application Ser. No. 12/396,605,filed Mar. 3, 2009 (which was a divisional of U.S. Pat. No. 7,527,787);U.S. Ser. No. 12/396,965, filed Mar. 3, 2009 (which was a divisional ofU.S. Pat. No. 7,521,056); U.S. Pat. Nos. 7,550,143 and 7,534,866; andU.S. Pat. No. 7,666,400. Those applications claimed the benefit under 35U.S.C. 119(e) of U.S. Provisional Patent Application Ser. Nos.60/668,603, filed Apr. 6, 2005; 60/728,292, filed Oct. 19, 2005;60/751,196, filed Dec. 16, 2005, 60/782,332, filed Mar. 14, 2006; and60/864,530, filed Nov. 6, 2006. This application claims the benefitunder 35 U.S.C. 119(e) of U.S. Provisional Patent Application Ser. Nos.61/168,657 and 61/168,668, filed Apr. 13, 2009. The entire text of eachpriority application is incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Mar. 31, 2010, is namedIBC124US.txt and is 13,788 bytes in size.

BACKGROUND

1. Field of the Invention

The present invention relates to the design and generation of multimericcytokines that retain in vitro activity and show enhanced in vivoefficacy. In preferred embodiments, the dock-and-lock (DNL) method isused to produce tetrameric cytokines that are anchored on a humanizedmonoclonal antibody or fragment thereof (MAb) by site-specificconjugation of a cytokine-based DDD moiety with a recombinant IgG, inwhich each of the two antibody heavy chains is fused to an AD moiety anddimers of the DDD moiety bind to each AD moiety. In a more preferredembodiment, the cytokine is erythropoietin, granulocytecolony-stimulating factor (G-CSF) or interferon-α2b (IFNα2b), althoughthe disclosed methods may be used to produce DNL constructs comprisingany tetrameric cytokine and antibody or fragment. In a most preferredembodiment, a humanized anti-CD20 MAb (hA20) is conjugated to IFNα2b toform a 20-2b DNL construct that comprises four copies of IFNα2b. Thecytokine-MAb constructs are of use to treat a variety of disease statesand show greater potency against target cells, such as tumors, than theparent MAb alone, the cytokine alone, a non-conjugated combination ofMAb and cytokine or cytokine conjugated to a control MAb.

2. Related Art

IFNα2b

Interferon-α (IFNα) has been reported to have anti-tumor activity inboth animal models of cancer (Ferrantini et al., 1994, J Immunol153:4604-15) and human cancer patients (Gutterman et al., 1980, AnnIntern Med 93:399-406). IFNα can exert a variety of direct anti-tumoreffects, including down-regulation of oncogenes, up-regulation of tumorsuppressors, enhancement of immune recognition via increased expressionof tumor surface MHC class I proteins, potentiation of apoptosis, andsensitization to chemotherapeutic agents (Gutterman et al., 1994, PNASUSA 91:1198-205; Matarrese et al., 2002, Am J Pathol 160:1507-20;Mecchia et al., 2000, Gene Ther 7:167-79; Sabaawy et al., 1999, Int JOncol 14:1143-51; Takaoka et al, 2003, Nature 424:516-23). For sometumors, IFNα can have a direct and potent anti-proliferative effectthrough activation of STAT1 (Grimley et al., 1998 Blood 91:3017-27).Indirectly, IFNα can inhibit angiogenesis (Sidky and Borden, 1987,Cancer Res 47:5155-61) and stimulate host immune cells, which may bevital to the overall antitumor response but has been largelyunder-appreciated (Belardelli et al., 1996, Immunol Today 17:369-72).IFNα has a pleiotropic influence on immune responses through effects onmyeloid cells (Raefsky et al, 1985, J Immunol 135:2507-12; Luft et al,1998, J Immunol 161:1947-53), T-cells (Carrero et al, 2006, J Exp Med203:933-40; Pilling et al., 1999, Eur J Immuol 29:1041-50), and B-cells(Le et al, 2001, Immunity 14:461-70). As an important modulator of theinnate immune system, IFNα induces the rapid differentiation andactivation of dendritic cells (Belardelli et al, 2004, Cancer Res64:6827-30; Paquette et al., 1998, J Leukoc biol 64:358-67; Santini etal., 2000, J Exp med 191:1777-88) and enhances the cytotoxicity,migration, cytokine production and antibody-dependent cellularcytotoxicity (ADCC) of NK cells (Biron et al., 1999, Annu Rev Immunol17:189-220; Brunda et al. 1984, Cancer Res 44:597-601).

The promise of IFNα as a cancer therapeutic has been hindered primarilydue to its short circulating half-life and systemic toxicity. PEGylatedforms of IFNα2 display increased circulation time, which augments theirbiological efficacy (Harris and Chess, 2003, Nat Rev Drug Discov2:214-21; Osborn et al., 2002, J Pharmacol Exp Ther 303:540-8). Fusionof IFNα to a monoclonal antibody (MAb) can provide similar benefits asPEGylation, including reduced renal clearance, improved solubility andstability, and markedly increased circulating half-life. The immediateclinical benefit of this is the requirement for less frequent and lowerdoses, allowing prolonged therapeutic concentrations. Targeting of IFNαto tumors using MAbs to a tumor-associated antigen (TAA) cansignificantly increase its tumor accretion and retention while limitingits systemic concentration, thereby increasing the therapeutic index.Increased tumor concentrations of IFNα can augment its directantiproliferative, apoptotic and anti-angiogenic activity, as well asprime and focus an antitumor immune response. Studies in mice usingsyngeneic murine IFNα-secreting transgenic tumors demonstrated anenhanced immune response elicited by a localized concentration of IFNα(Ferrantini et al., 2007, Biochimie 89:884-93).

G-CSF

Granulocyte colony-stimulating factor (G-CSF) stimulates the productionof neutrophils and regulates the survival, proliferation, anddifferentiation of hematopoietic progenitors. In the U.S., a recombinantmethionyl human G-CSF and its longer-acting PEGylated form are usedprimarily for treating chemotherapy-induced neutropenia to directlyincrease neutrophil counts and for mobilizing transplantable stem cellsfrom bone marrow to the blood for easier collection and processing.

Erythropoietin

Erythropoietin (Epo) is a hematopoietic growth factor that stimulatesthe proliferation and differentiation of erythrocytes into mature redblood cells. Several recombinant human Epo are currently used for thetreatment of anemia, predominantly associated with chronic renal failureand post cancer chemotherapy. However, the short half-life (4-13 hours)of rhEpo necessitates frequent dosing and, therefore, increasing theserum half-life of Epo to allow less frequent dosing is highly desirableand has been an important goal for developing next-generation Epo.

Each of the cytokines discussed above, as well as many other cytokines,would benefit from methods and compositions to provide prolongedbioavailability and/or targeting of cytokine to specific cell types,according to the methods and compositions of the instant invention.

SUMMARY OF THE INVENTION

The present invention discloses methods and compositions for conjugatesof cytokines and antibodies, prepared using the Dock-and-Lock (DNL)method (Chang et al., 2007, Clin Cancer Res 13:5586s-91s), whichgenerates stable and defined conjugates suitable for in vivoapplications. In preferred embodiments, the DNL complexes comprise fourcopies of a cytokine, such as IFNα2b, G-CSF or Epo, each attached to adimerization and docking domain (DDD) moiety. The DDD moietiesspontaneously dimerize and each DDD dimer binds to an anchor domain (ADmoiety). In more preferred embodiments, an AD moiety is attached to theC-terminal end of each heavy chain constant region of an IgG antibody,resulting in a tetrameric cytokine-IgG DNL complex.

However, the skilled artisan will realize that other types of DNLcomplexes with different structures and different ratios of cytokine toantibody or antibody fragment may be constructed and used within thescope of the claimed methods and compositions, such as those disclosedin U.S. Pat. Nos. 7,550,143; 7,521,056; 7,534,866; 7,527,787 and7,666,400, the Examples section of each of which is incorporated hereinby reference. For convenience, the DNL constructs of interest aregenerally referred to herein as cytokine-MAb constructs. However, theskilled artisan will realize that the designation “MAb” is used as ageneric term to indicate the presence of an antibody or antigen-bindingfragment thereof.

The skilled artisan will further realize that any known antibody orantigen-binding fragment thereof may be incorporated into thecytokine-MAb DNL constructs. In certain embodiments, the complex is ofuse for cancer therapy and the antibody binds to a tumor associatedantigen (TAA). A variety of tumor-associated antigens are known in theart, including but not limited to carbonic anhydrase IX, CCCL19, CCCL21,CSAp, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18,CD19, IGF-1R, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33,CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD55, CD59, CD64,CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138,CD147, CD154, AFP, PSMA, CEACAM5, CEACAM-6, B7, ED-B of fibronectin,Factor H, FHL-1, Flt-3, folate receptor, GROB, HMGB-1, hypoxia induciblefactor (HIF), HM1.24, insulin-like growth factor-1 (ILGF-1), IFN-γ,IFN-α, IFN-β, IL-2, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6,IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, IP-10, MAGE, mCRP, MCP-1,MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5, PAM4 antigen, NCA-95,NCA-90, Ia, HM1.24, EGP-1, EGP-2, HLA-DR, tenascin, Le(y), RANTES, T101,TAC, Tn antigen, Thomson-Friedenreich antigens, tumor necrosis antigens,TNF-α, TRAIL receptor (R1 and R2), VEGFR, EGFR, P1GF, complement factorsC3, C3a, C3b, C5a, C5, and an oncogene product.

Exemplary anti-cancer antibodies that may be utilized in thecytokine-MAb DNL constructs include, but are not limited to, hR1(anti-IGF-1R, U.S. patent application Ser. No. 12/722,645, filed Feb.12, 2010) hPAM4 (anti-mucin, U.S. Pat. No. 7,282,567), hA20 (anti-CD20,U.S. Pat. No. 7,251,164), hA19 (anti-CD19, U.S. Pat. No. 7,109,304),hIMMU31 (anti-AFP, U.S. Pat. No. 7,300,655), hLL1 (anti-CD74, U.S. Pat.No. 7,312,318), hLL2 (anti-CD22, U.S. Pat. No. 7,074,403), hMu-9(anti-CSAp, U.S. Pat. No. 7,387,773), hL243 (anti-HLA-DR, U.S. Pat. No.7,612,180), hMN-14 (anti-CEA, U.S. Pat. No. 6,676,924), hMN-15(anti-CEA, U.S. Pat. No. 7,541,440), hRS7 (anti-EGP-1, U.S. Pat. No.7,238,785) and hMN-3 (anti-CEA, U.S. Pat. No. 7,541,440) the Examplessection of each cited patent or application incorporated herein byreference. The skilled artisan will realize that this list is notlimiting and any other known anti-TAA antibody may be incorporated intothe cytokine-MAb DNL constructs.

In certain preferred embodiments, the target antigen may be CD20, whichis an attractive candidate TAA for the therapy of B-cell lymphomas andother hematopoietic disorders. Anti-CD20 immunotherapy with rituximab isone of the most successful therapies against lymphoma, with relativelylow toxicity (McLaughlin et al., 1998, J Clin Oncol 16:2825-33). Sincerituximab is a chimeric antibody that can show immunogenicity in somepatient populations and has considerably long infusion times for theinitial administration (Cheson et al., 2008, NEJM 359:613-26), a bettercandidate for CD20-targeting is the humanized MAb, veltuzumab (Stein etal., 2004, Clin Cancer Res 10:2868-78). Combination therapies withrituximab and IFNα currently under clinical evaluation have shownimproved efficacy over rituximab alone (Kimby et al., 2008, LeukLymphoma 49:102-12; Salles et al., 2008, Blood 112:4824-31). Thesestudies demonstrate some advantages of this combination as well as thedrawbacks associated with IFNα. In addition to weekly infusions withrituximab, patients are typically administered IFNα three times/week formonths and suffer the flu-like symptoms that are common side effectsassociated with IFNα therapy and which limit the tolerable dose. Ananti-CD20 MAb-IFNα conjugate could allow the less frequentadministration of a single agent at a lower dose, limit or eliminateside effects, and may result in far superior efficacy.

Other types of target antigen are of use for antibody-based therapy ofdifferent disease states and cytokine-MAb DNL constructs incorporatingantibodies that target any such alternative antigen may be utilized inthe claimed methods and compositions.

Exemplary cytokines that may be incorporated into the cytokine-MAb DNLconstructs include but are not limited to MIF (macrophage migrationinhibitory factor), HMGB-1 (high mobility group box protein 1), TNF-α,IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-19, IL-23, IL-24, CCL19,CCL21, IL-8, MCP-1, RANTES, MIP-1A, MIP-1B, ENA-78, MCP-1, IP-10, Gro-β,Eotaxin, interferon-α, -β, -λ, G-CSF, GM-CSF, SCF, PDGF, MSF, Flt-3ligand, erythropoietin, thrombopoietin, CNTF, leptin, oncostatin M,VEGF, EGF, FGF, P1GF, insulin, hGH, calcitonin, Factor VIII, IGF,somatostatin, tissue plasminogen activator and LIF. As discussed below,the amino acid sequences of a wide variety of human cytokines are knownin the art and may be obtained, for example, from the NCBI proteinsequence database and other publicly known sources.

Certain embodiments may concern therapeutic or diagnostic conjugates ofcytokine-MAb DNL constructs, bound to at least one therapeutic agent orat least one diagnostic agent. Constructs with multiple therapeuticagents of the same or different type are also encompassed.Alternatively, the cytokine-MAb DNL constructs may be administered incombination with at least one therapeutic agent administered before,simultaneously with or after the cytokine-MAb construct. Any therapeuticagent known in the art, as discussed in more detail below, may beutilized in combination with or attached to cytokine-MAb DNL construct,including but not limited to radionuclides, immunomodulators,anti-angiogenic agents, cytokines, chemokines, growth factors, hormones,drugs, prodrugs, enzymes, oligonucleotides, siRNAs, pro-apoptoticagents, photoactive therapeutic agents, cytotoxic agents,chemotherapeutic agents, toxins, other antibodies or antigen bindingfragments thereof.

The cytokine-MAb complexes are of use for therapy of a wide variety ofdiseases or medical conditions, including but not limited to cancer,autoimmune disease, hyperplasia, septicemia, diabetes, inflammatorybowel disease, Crohn's disease, ulcerative colitis, rheumatoidarthritis, sarcoidosis, asthma, anemia, neutropenia and Osler-WebberSyndrome.

In particular embodiments, the disclosed methods and compositions may beof use to treat autoimmune disease, such as acute idiopathicthrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura,dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupuserythematosus, lupus nephritis, rheumatic fever, polyglandularsyndromes, bullous pemphigoid, juvenile diabetes mellitus,Henoch-Schonlein purpura, post-streptococcal nephritis, erythemanodosurn, Takayasu's arteritis, Addison's disease, rheumatoid arthritis,multiple sclerosis, sarcoidosis, ulcerative colitis, erythemamultiforme, IgA nephropathy, polyarteritis nodosa, ankylosingspondylitis, Goodpasture's syndrome, thromboangitis obliterans,Sjogren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris,Wegener's granulomatosis, membranous nephropathy, amyotrophic lateralsclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, perniciousanemia, rapidly progressive glomerulonephritis, psoriasis or fibrosingalveolitis.

Exemplary types of cancers that may be treated with the cytokine-MAb DNLconstructs include but are not limited to acute lymphoblastic leukemia,acute myelogenous leukemia, biliary cancer, breast cancer, cervicalcancer, chronic lymphocytic leukemia, chronic myelogenous leukemia,colorectal cancer, endometrial cancer, esophageal cancer, gastriccancer, head and neck cancer, Hodgkin's lymphoma, lung cancer, medullarythyroid cancer, non-Hodgkin's lymphoma, multiple myeloma, renal cancer,ovarian cancer, pancreatic cancer, glioma, melanoma, liver cancer,prostate cancer, urinary bladder cancer, Wilms' tumor, Ewing sarcoma,neuroendocrine tumors, glioblastomas, neuroblastoma, melanoma, skincancer, liver cancer, medullary thyroid carcinoma.

Still other embodiments relate to DNA sequences encoding fusionproteins, such as cytokine-DDD fusion proteins or Fab-AD fusionproteins, vectors and host cells containing the DNA sequences, andmethods of making the cytokine-MAb DNL constructs.

A particularly preferred embodiment concerns 20-2b, an IFNα-MAb DNLconstruct comprising four IFNα2b groups attached to a humanizedanti-CD20 antibody (hA20). The 20-2b construct was found to be asuperior anti-lymphoma agent to either the parent antibody alone, theIFNα cytokine alone, or a non-targeting IFNα-MAb by in vitroproliferation, ex vivo lymphoma cell depletion, and in vivo therapystudies. The 20-2b construct is of particular use for therapy oflymphomas, leukemias, myelomas and related conditions.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the terms “a”, “an” and “the” may refer to either thesingular or plural, unless the context otherwise makes clear that onlythe singular is meant.

As used herein, the term “about” means plus or minus ten percent (10%)of a value. For example, “about 100” would refer to any number between90 and 110.

An antibody refers to a full-length (i.e., naturally occurring or formedby normal immunoglobulin gene fragment recombinatorial processes)immunoglobulin molecule (e.g., an IgG antibody) or an immunologicallyactive, antigen-binding portion of an immunoglobulin molecule, like anantibody fragment.

An antibody fragment is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, Fv, scFv and the like. Regardless of structure, anantibody fragment binds with the same antigen that is recognized by theintact antibody. The term “antibody fragment” also includes isolatedfragments consisting of the variable regions, such as the “Fv” fragmentsconsisting of the variable regions of the heavy and light chains andrecombinant single chain polypeptide molecules in which light and heavyvariable regions are connected by a peptide linker (“scFv proteins”). Asused herein, the term “antibody fragment” does not include portions ofantibodies without antigen binding activity, such as Fc fragments orsingle amino acid residues.

The term antibody fusion protein may refer to a recombinantly producedantigen-binding molecule in which one or more of the same or differentsingle-chain antibody or antibody fragment segments with the same ordifferent specificities are linked. Valency of the fusion proteinindicates how many binding arms or sites the fusion protein has to asingle antigen or epitope; i.e., monovalent, bivalent, trivalent ormultivalent. The multivalency of the antibody fusion protein means thatit can take advantage of multiple interactions in binding to an antigen,thus increasing the avidity of binding to the antigen. Specificityindicates how many antigens or epitopes an antibody fusion protein isable to bind; i.e., monospecific, bispecific, trispecific,multispecific. Using these definitions, a natural antibody, e.g., anIgG, is bivalent because it has two binding arms but is monospecificbecause it binds to one epitope. Monospecific, multivalent fusionproteins have more than one binding site for an epitope but only bindwith one epitope. The fusion protein may comprise a single antibodycomponent, a multivalent or multispecific combination of differentantibody components or multiple copies of the same antibody component.The fusion protein may additionally comprise an antibody or an antibodyfragment and a therapeutic agent, such as a cytokine. Other examples oftherapeutic agents suitable for such fusion proteins includeimmunomodulators and toxins. One preferred toxin comprises aribonuclease (RNase), preferably a recombinant RNase. However, the termis not limiting and a variety of protein or peptide effectors may beincorporated into a fusion protein. In another non-limiting example, afusion protein may comprise an AD or DDD sequence for producing a DNLconstruct as discussed below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cartoon drawing depicting the gene structures (A and B) forexpression of Cytokine-DDD2 (C), and IgG-AD2 (D) DNL modules. Themodules are combined to form DNL structures consisting of four cytokinesfused to an IgG (E).

FIG. 2 shows in vitro IFNα activity in a cytokine-MAb DNL constructcompared to PEGylated or native IFNα. Specific activities (IU/pmol)measured as described in the Examples. The activity of knownconcentrations of each test article was extrapolated from a rhIFNα2bstandard curve. Cultures were grown in the presence of increasingconcentrations of 20-2b (●), 734-2b (▪), v-mab (◯), v-mab+734-2b (□),PEGASYS® (▾), PEG-INTRON® (▴) or 1R-2b (∇) and the relative viable celldensities were measured with MTS. The % of the signal obtained fromuntreated cells was plotted vs. the log of the molar concentration.Dose-response curves and EC₅₀ values were generated using Prismsoftware. Error bars, SD.

FIG. 2(A) cell-based reporter gene assay.

FIG. 2(B) viral protection assay with EMC virus and A549 cells.

FIG. 2(C) In vitro lymphoma proliferation assays using Daudi cells.

FIG. 2(D) In vitro lymphoma proliferation assays using Jeko-1 cells.

FIG. 3 shows the results of pharmacokinetic analyses in Swiss-Webstermice. Mice were administered 20-2b, α2b-413, PEGINTRON® or PEGASYS® andserum samples were analyzed for IFNα2b concentration by ELISA over 96hours. Serum elimination curves are shown. Serum half-life (T_(1/2))elimination rates and mean residence times (MRT) are summarized in theinserted table.

FIG. 4(A) illustrates ADCC effector functions of 20-2b. Daudi or Rajicells were incubated with 20-2b, 22-2b, v-mab, epratuzumab (e-mab), orh734 at 5 μg/ml in the presence of freshly isolated PBMCs for 4 h beforequantification of cell lysis.

FIG. 4(B) shows CDC effector functions of 20-2B. Daudi cells wereincubated with serial dilutions of 20-2b (●), 734-2b (▪) or v-mab (◯) inthe presence of human complement. The % complement control (number ofviable cells in the test sample compared to cells treated withcomplement only) was plotted vs. the log of the nM concentration. Errorbars, SD.

FIG. 5 shows enhanced depletion of NHL cells from whole blood by 20-2b.Fresh heparinized human blood was mixed with either Daudi or Ramos andincubated with 20-2b (●), v-mab (◯), 734-2b (▪) or v-mab+734-2b (□) at0.01, 0.1 or 1 nM for two days. The effect of the indicated treatmentson lymphoma and peripheral blood lymphocytes was evaluated using flowcytometry. Error bars, SD.

FIG. 6(A) illustrates survival curves showing therapeutic efficacy of20-2b in a disseminated Burkitt's lymphoma (Daudi) xenograft model.Female C.B. 17 SCID mice were administered Daudi cells i.v. on day 0.Treatments consisted of 20-2b (●), 734-2b (▪), v-mab (◯), PEGASYS® (▾)or saline (X) given as a single s.c. doses. Days of treatment areindicated with arrows. Survival curves were analyzed using Prismsoftware. In an Early Daudi model. Groups of 10 mice were given a singledose of 0.7 pmol (solid line) or 0.07 pmol (dashed line) on day 1.

FIG. 6(B) shows a similar study to FIG. 6(A), but in an Advanced Daudimodel. Groups of 10 mice were given a single dose of 0.7 pmol (solidline), 7 pmol (dashed line) or 70 pmol (gray line) on day 7.

FIG. 7(A) presents survival curves showing therapeutic efficacy of 20-2bin disseminated Burkitt's lymphoma (Raji and NAMALWA) xenograft models.Female C.B. 17 SCID mice were administered NHL cells i.v. on day 0.Treatments consisted of 20-2b (●), 734-2b (▪), v-mab (◯) or saline (X)given as s.c. doses. Days of treatment are indicated with arrows.Survival curves were analyzed using Prism software. In an Advanced Rajimodel, groups of 10 received 250 pmol doses on days 5, 7, 9, 12, 14 and16.

FIG. 7(B) shows a similar study to FIG. 7(A), but in an Early NAMALWAmodel. Groups of 6 received 250 pmol doses of 20-2b or 734-2b on days 1,3, 5, 8, 10 and 12 or 3.5 nmol doses of v-mab on days 1, 5, 9, 13, 17,21 and 25.

FIG. 8 shows the results of a cell-based assay for EPO activity usingTF1 cells that were treated with EPO standard, 734-EPO, or EPO-DDD2 for72 hours. Dose response curves and EC₅₀ values were generated usingGraph Pad Prism software.

FIG. 9. In vitro proliferation assay with tetrameric G-CSF DNL constructin Kasumi-1 myeloid leukemia cells, showing that IgG-G-CSF complexes aremore potent than recombinant hG-CSF.

FIG. 10. ADCC activity of hA20-G-CSF DNL construct evaluated inCD20-positive Daudi cells with the PBMCs isolated from two differentdonors. For both donors, enhanced ADCC was observed for hA20-G-CSFcompared to the parental hA20 IgG (veltuzumab): donor 006, 35% vs. 20%(P<0.0191); donor 010, 60% vs. 50% (not statistically significant). Theconcentration of each agent tested was 5 μg/mL.

FIG. 11. In vivo activity of G-CSF DNL construct.

FIG. 12. In vitro potency of Epo DNL constructs, measured by theirability to stimulate proliferation of the cytokine-dependent TF1 cellline. TF1 cells were incubated with serial dilutions of rhEpo, ARANESP®,Epo-DDD2, Fab-Epo, IgG-Epo or 457-Epo. The results show that theEpo-DDD2 module and each DNL construct maintained Epo biologicalactivity with similar specific activity as ARANESP®.

FIG. 13. In vivo activity of Epo DNL construct. Normal Swiss-Webstermice (n=4) were administered a single i.v. injection (3.2 μg) of IgG-Epoand the hematocrit was measured after five days. The results show thattreatment with IgG-Epo produced a significant (P=0.0107) increase inhematocrit compared to untreated mice.

Dock and Lock (DNL) Method

The DNL method exploits specific protein/protein interactions that occurbetween the regulatory (R) subunits of cAMP-dependent protein kinase(PKA) and the anchoring domain (AD) of A-kinase anchoring proteins(AKAPs) (Baillie et al., FEBS Letters. 2005; 579: 3264. Wong and Scott,Nat. Rev. Mol. Cell. Biol. 2004; 5: 959). PKA, which plays a centralrole in one of the best studied signal transduction pathways triggeredby the binding of the second messenger cAMP to the R subunits, was firstisolated from rabbit skeletal muscle in 1968 (Walsh et al., J. Biol.Chem. 1968; 243:3763). The structure of the holoenzyme consists of twocatalytic subunits held in an inactive form by the R subunits (Taylor,J. Biol. Chem. 1989; 264:8443). Isozymes of PKA are found with two typesof R subunits (RI and RII), and each type has a and β isoforms (Scott,Pharmacol. Ther. 1991; 50:123). The R subunits have been isolated onlyas stable dimers and the dimerization domain has been shown to consistof the first 44 amino-terminal residues (Newlon et al., Nat. Struct.Biol. 1999; 6:222). Binding of cAMP to the R subunits leads to therelease of active catalytic subunits for a broad spectrum ofserine/threonine kinase activities, which are oriented toward selectedsubstrates through the compartmentalization of PKA via its docking withAKAPs (Scott et al., J. Biol. Chem. 1990; 265; 21561)

Since the first AKAP, microtubule-associated protein-2, wascharacterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci. USA. 1984;81:6723), more than 50 AKAPs that localize to various sub-cellularsites, including plasma membrane, actin cytoskeleton, nucleus,mitochondria, and endoplasmic reticulum, have been identified withdiverse structures in species ranging from yeast to humans (Wong andScott, Nat. Rev. Mol. Cell. Biol. 2004; 5:959). The AD of AKAPs for PKAis an amphipathic helix of 14-18 residues (Carr et al., J. Biol. Chem.1991; 266:14188). The amino acid sequences of the AD are quite variedamong individual AKAPs, with the binding affinities reported for RIIdimers ranging from 2 to 90 nM (Alto et al., Proc. Natl. Acad. Sci. USA.2003; 100:4445). Interestingly, AKAPs will only bind to dimeric Rsubunits. For human RIIα, the AD binds to a hydrophobic surface formedby the 23 amino-terminal residues (Colledge and Scott, Trends Cell Biol.1999; 6:216). Thus, the dimerization domain and AKAP binding domain ofhuman RIIα are both located within the same N-terminal 44 amino acidsequence (Newlon et al., Nat. Struct. Biol. 1999; 6:222; Newlon et al.,EMBO J. 2001; 20:1651), which is termed the DDD herein.

DDD of Human RIIα and AD of AKAPs as Linker Modules

We have developed a platform technology to utilize the DDD of human RIIαand the AD of AKAP proteins as an excellent pair of linker modules fordocking any two entities, referred to hereafter as A and B, into anoncovalent complex, which could be further locked into a stablytethered structure through the introduction of cysteine residues intoboth the DDD and AD at strategic positions to facilitate the formationof disulfide bonds. The general methodology of the “dock-and-lock”approach is as follows. Entity A is constructed by linking a DDDsequence to a precursor of A, resulting in a first component hereafterreferred to as a. Because the DDD sequence would effect the spontaneousformation of a dimer, A would thus be composed of a₂. Entity B isconstructed by linking an AD sequence to a precursor of B, resulting ina second component hereafter referred to as b. The dimeric motif of DDDcontained in a₂ will create a docking site for binding to the ADsequence contained in b, thus facilitating a ready association of a₂ andb to form a binary, trimeric complex composed of a₂b. This binding eventis made irreversible with a subsequent reaction to covalently secure thetwo entities via disulfide bridges, which occurs very efficiently basedon the principle of effective local concentration because the initialbinding interactions should bring the reactive thiol groups placed ontoboth the DDD and AD into proximity (Chmura et al., Proc. Natl. Acad.Sci. USA. 2001; 98:8480) to ligate site-specifically.

In preferred embodiments, the cytokine-MAb DNL constructs are based on avariation of the a₂b structure, in which an IgG immunoglobulin moleculeis attached at its C-terminal end to two copies of an AD moiety. Each ADmoiety is capable of binding to two DDD moieties in the form of a dimer.By attaching a cytokine to each DDD moiety, four copies of cytokine areconjugated to each IgG molecule. In more preferred embodiments, each ofthe four cytokines in a DNL construct is identical.

By attaching the DDD and AD away from the functional groups of the twoprecursors, such site-specific ligations are also expected to preservethe original activities of the two precursors. This approach is modularin nature and potentially can be applied to link, site-specifically andcovalently, a wide range of substances. The DNL method was disclosed inU.S. Pat. Nos. 7,550,143; 7,521,056; 7,534,866; 7,527,787 and 7,666,400.

In preferred embodiments, as illustrated in the Examples below, theeffector moiety is a protein or peptide, more preferably an antibody,antibody fragment or cytokine, which can be linked to a DDD or AD unitto form a fusion protein or peptide. A variety of methods are known formaking fusion proteins, including nucleic acid synthesis, hybridizationand/or amplification to produce a synthetic double-stranded nucleic acidencoding a fusion protein of interest. Such double-stranded nucleicacids may be inserted into expression vectors for fusion proteinproduction by standard molecular biology techniques (see, e.g. Sambrooket al., Molecular Cloning, A laboratory manual, 2^(nd) Ed, 1989). Insuch preferred embodiments, the AD and/or DDD moiety may be attached toeither the N-terminal or C-terminal end of an effector protein orpeptide. However, the skilled artisan will realize that the site ofattachment of an AD or DDD moiety to an effector moiety may vary,depending on the chemical nature of the effector moiety and the part(s)of the effector moiety involved in its physiological activity.Site-specific attachment of a variety of effector moieties may beperformed using techniques known in the art, such as the use of bivalentcross-linking reagents and/or other chemical conjugation techniques.

DDD and AD Sequence Variants

In certain embodiments, the AD and DDD sequences incorporated into thecytokine-PEG DNL complex comprise the amino acid sequences of DDD1 (SEQID NO:1) and AD1 (SEQ ID NO:3) below. In more preferred embodiments, theAD and DDD sequences comprise the amino acid sequences of DDD2 (SEQ IDNO:2) and AD2 (SEQ ID NO:4), which are designed to promote disulfidebond formation between the DDD and AD moieties.

DDD1 (SEQ ID NO: 1) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA DDD2(SEQ ID NO: 2) CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA AD1(SEQ ID NO: 3) QIEYLAKQIVDNAIQQA AD2 (SEQ ID NO: 4)CGQIEYLAKQIVDNAIQQAGC

However, in alternative embodiments sequence variants AD and/or DDDmoieties may be utilized in construction of the cytokine-PEG DNLcomplexes. The structure-function relationships of the AD and DDDdomains have been the subject of investigation. (See, e.g., Burns-Hamuroet al., 2005, Protein Sci 14:2982-92; Carr et al., 2001, J Biol Chem276:17332-38; Alto et al., 2003, Proc Natl Acad Sci USA 100:4445-50;Hundsrucker et al., 2006, Biochem J 396:297-306; Stokka et al., 2006,Biochem J 400:493-99; Gold et al., 2006, Mol Cell 24:383-95; Kindermanet al., 2006, Mol Cell 24:397-408, the entire text of each of which isincorporated herein by reference.)

For example, Kinderman et al. (2006) examined the crystal structure ofthe AD-DDD binding interaction and concluded that the human DDD sequencecontained a number of conserved amino acid residues that were importantin either dimer formation or AKAP binding, underlined below in SEQ IDNO:1. (See FIG. 1 of Kinderman et al., 2006, incorporated herein byreference.) The skilled artisan will realize that in designing sequencevariants of the DDD sequence, one would desirably avoid changing any ofthe underlined residues, while conservative amino acid substitutionsmight be made for residues that are less critical for dimerization andAKAP binding. Thus, a potential alternative DDD sequence of use forconstruction of DNL complexes is shown in SEQ ID NO:5, wherein “X”represents a conservative amino acid substitution. Conservative aminoacid substitutions are discussed in more detail below, but could involvefor example substitution of an aspartate residue for a glutamateresidue, or a leucine or valine residue for an isoleucine residue, etc.Such conservative amino acid substitutions are well known in the art.

Human DDD Sequence from Protein Kinase A

(SEQ ID NO: 1) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 5) XXIXIXXXLXXLLXXYXVXVLXXXXXXLVXFXVXYFXXLXXXXX 

Alto et al. (2003) performed a bioinformatic analysis of the AD sequenceof various AKAP proteins to design an RII selective AD sequence calledAKAP-IS (SEQ ID NO:3), with a binding constant for DDD of 0.4 nM. TheAKAP-IS sequence was designed as a peptide antagonist of AKAP binding toPKA. Residues in the AKAP-IS sequence where substitutions tended todecrease binding to DDD are underlined in SEQ ID NO:3. Therefore, theskilled artisan will realize that variants which may function for DNLconstructs are indicated by SEQ ID NO:6, where “X” is a conservativeamino acid substitution.

AKAP-IS sequence QIEYLAKQIVDNAIQQA  (SEQ ID NO: 3) XXXXXAXXIVXXAIXXX(SEQ ID NO: 6)

Similarly, Gold (2006) utilized crystallography and peptide screening todevelop a SuperAKAP-IS sequence (SEQ ID NO:7), exhibiting a five orderof magnitude higher selectivity for the RII isoform of PKA compared withthe RI isoform. Underlined residues indicate the positions of amino acidsubstitutions, relative to the AKAP-IS sequence, that increased bindingto the DDD moiety of RIIα. In this sequence, the N-terminal Q residue isnumbered as residue number 4 and the C-terminal A residue is residuenumber 20. Residues where substitutions could be made to affect theaffinity for RIIα were residues 8, 11, 15, 16, 18, 19 and 20 (Gold etal., 2006). It is contemplated that in certain alternative embodiments,the SuperAKAP-IS sequence may be substituted for the AKAP-IS AD moietysequence to prepare cytokine-PEG DNL constructs. Other alternativesequences that might be substituted for the AKAP-IS AD sequence areshown in SEQ ID NO:8-10. Substitutions relative to the AKAP-IS sequenceare underlined. It is anticipated that, as with the AKAP-IS sequenceshown in SEQ ID NO:3, the AD moiety may also include the additionalN-terminal residues cysteine and glycine and C-terminal residues glycineand cysteine, as shown in SEQ ID NO:4.

SuperAKAP-IS QIEYVAKQIVDYAIHQA (SEQ ID NO: 7) Alternative AKAP sequencesQIEYKAKQIVDHAIHQA  (SEQ ID NO: 8) QIEYHAKQIVDHAIHQA  (SEQ ID NO: 9)QIEYVAKQIVDHAIHQA  (SEQ ID NO: 10)

Stokka et al. (2006) also developed peptide competitors of AKAP bindingto PKA, shown in SEQ ID NO:11-13. The peptide antagonists weredesignated as Ht31 (SEQ ID NO:11), RIAD (SEQ ID NO:12) and PV-38 (SEQ IDNO:13). The Ht-31 peptide exhibited a greater affinity for the RIIisoform of PKA, while the RIAD and PV-38 showed higher affinity for RI.

Ht31 DLIEEAASRIVDAVIEQVKAAGAY (SEQ ID NO: 11) RIAD LEQYANQLADQIIKEATE(SEQ ID NO: 12) PV-38 FEELAWKIAKMIWSDVFQQC  (SEQ ID NO: 13)

Hundsrucker et al. (2006) developed still other peptide competitors forAKAP binding to PKA, with a binding constant as low as 0.4 nM to the DDDof the RII form of PKA. The sequences of various AKAP antagonisticpeptides is provided in Table 1 of Hundsrucker et al. (incorporatedherein by reference). Residues that were highly conserved among the ADdomains of different AKAP proteins are indicated below by underliningwith reference to the AKAP IS sequence (SEQ ID NO:3). The residues arethe same as observed by Alto et al. (2003), with the addition of theC-terminal alanine residue. (See FIG. 4 of Hundsrucker et al. (2006),incorporated herein by reference.) The sequences of peptide antagonistswith particularly high affinities for the RII DDD sequence are shown inSEQ ID NO:14-16.

AKAP-IS QIEYLAKQIVDNAIQQA (SEQ ID NO: 3) AKAP7δ-wt-pepPEDAELVRLSKRLVENAVLKAVQQY (SEQ ID NO: 14) AKAP7δ-L304T-pepPEDAELVRTSKRLVENAVLKAVQQY (SEQ ID NO: 15) AKAP7δ-L308D-pepPEDAELVRLSKRDVENAVLKAVQQY (SEQ ID NO: 16)

Carr et al. (2001) examined the degree of sequence homology betweendifferent AKAP-binding DDD sequences from human and non-human proteinsand identified residues in the DDD sequences that appeared to be themost highly conserved among different DDD moieties. These are indicatedbelow by underlining with reference to the human PKA RIIα DDD sequenceof SEQ ID NO:2. Residues that were particularly conserved are furtherindicated by italics. The residues overlap with, but are not identicalto those suggested by Kinderman et al. (2006) to be important forbinding to AKAP proteins. Thus, a potential DDD sequence is indicated inSEQ ID NO:17, wherein “X” represents a conservative amino acidsubstitution.

(SEQ ID NO: 1) SHIQ IP P GL TELLQGYT V EVLR Q QP P DLVEFA VE YF TR L REAR A  (SEQ ID NO: 17) XHIX IP X GL XELLQGYT X EVLR X QP X DLVEFA XX YF XXL XEX R X 

The skilled artisan will realize that in general, those amino acidresidues that are highly conserved in the DDD and AD sequences fromdifferent proteins are ones that it may be preferred to remain constantin making amino acid substitutions, while residues that are less highlyconserved may be more easily varied to produce sequence variants of theAD and/or DDD sequences described herein.

In addition to sequence variants of the DDD and/or AD moieties, incertain embodiments it may be preferred to introduce sequence variationsin the antibody moiety or the linker peptide sequence joining theantibody with the AD sequence. In one illustrative example, threepossible variants of fusion protein sequences, are shown in SEQ IDNO:18-20.

(L) QKSLSLSPGLGSGGGGSGGCG  (SEQ ID NO: 18) (A) QKSLSLSPGAGSGGGGSGGCG(SEQ ID NO: 19) (-) QKSLSLSPGGSGGGGSGGCG (SEQ ID NO: 20)Cytokines and Other Immunomodulators

In certain preferred embodiments, the effector moiety is animmunomodulator. An immunomodulator is an agent that when present,alters, suppresses or stimulates the body's immune system.Immunomodulators of use may include a cytokine, a stem cell growthfactor, a lymphotoxin, an hematopoietic factor, a colony stimulatingfactor (CSF), an interferon (IFN), erythropoietin, thrombopoietin and acombination thereof. Specifically useful are lymphotoxins such as tumornecrosis factor (TNF), hematopoietic factors, such as interleukin (IL),colony stimulating factor, such as granulocyte-colony stimulating factor(G-CSF) or granulocyte macrophage-colony stimulating factor (GM-CSF),interferon, such as interferons-α, -β or -γ, and stem cell growthfactor, such as that designated “S1 factor”.

In more preferred embodiments, the effector moieties are cytokines, suchas lymphokines, monokines, growth factors and traditional polypeptidehormones. Included among the cytokines are growth hormones such as humangrowth hormone, N-methionyl human growth hormone, and bovine growthhormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin;prorelaxin; glycoprotein hormones such as follicle stimulating hormone(FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH);placenta growth factor (P1GF), hepatic growth factor; prostaglandin,fibroblast growth factor; prolactin; placental lactogen, OB protein;tumor necrosis factor-α and -β; mullerian-inhibiting substance; mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor; integrin; thrombopoietin (TPO); nerve growth factors suchas NGF-β; platelet-growth factor; transforming growth factors (TGFs)such as TGF-α and TGF-β; insulin-like growth factor-I and -II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-α, -β, and -γ; colony stimulating factors (CSFs) such asmacrophage-CSF (M-CSF); interleukins (ILs) such as IL-1, IL-1α, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13,IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, kit-ligand or FLT-3,angiostatin, thrombospondin, endostatin, tumor necrosis factor (TNF,such as TNF-α) and LT.

The amino acid sequences of protein or peptide immunomodulators, such ascytokines, are well known in the art and any such known sequences may beused in the practice of the instant invention. The skilled artisan isaware of numerous sources of public information on cytokine sequence.For example, the NCBI database contains both protein and encodingnucleic acid sequences for a large number of cytokines andimmunomodulators, such as erythropoietin (GenBank NM 000799), IL-1 beta(GenPept AAH08678), GM-CSF (GenPept AAA52578), G-CSF (GenPept CAA27290),TNF-α(GenPept CAA26669), interferon-alpha (GenPept AAA52716.1),interferon-alpha 2b (GenPept AAP20099.1) and virtually any of thepeptide or protein immunomodulators listed above. It is a matter ofroutine for the skilled artisan to identify an appropriate amino acidand/or nucleic acid sequence for essentially any protein or peptideeffector moiety of interest.

Antibodies and Antibody Fragments

In various embodiments, antibodies or antigen-binding fragments ofantibodies may be attached to the multimeric cytokines. Antigen-bindingantibody fragments are well known in the art, such as F(ab′)₂, F(ab)₂,Fab′, Fab, Fv, scFv and the like, and any such known fragment may beused. As used herein, an antigen-binding antibody fragment refers to anyfragment of an antibody that binds with the same antigen that isrecognized by the intact or parent antibody. Techniques for preparing ADand/or DDD conjugates of virtually any antibody or fragment of interestare known (e.g., U.S. Pat. Nos. 7,550,143; 7,521,056; 76,534,866;7,527,787 and 7,666,400).

An antibody or fragment thereof may be used which is not conjugated to atherapeutic agent—referred to as a “naked” antibody or fragment thereof.In alternative embodiments, antibodies or fragments may be conjugated toone or more therapeutic and/or diagnostic agents. A wide variety of suchtherapeutic and diagnostic agents are known in the art, as discussed inmore detail below, and any such known therapeutic or diagnostic agentmay be used.

Techniques for preparing monoclonal antibodies against virtually anytarget antigen are well known in the art. See, for example, Kohler andMilstein, Nature 256: 495 (1975), and Coligan et al. (eds.), CURRENTPROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons1991). Briefly, monoclonal antibodies can be obtained by injecting micewith a composition comprising an antigen, removing the spleen to obtainB-lymphocytes, fusing the B-lymphocytes with myeloma cells to producehybridomas, cloning the hybridomas, selecting positive clones whichproduce antibodies to the antigen, culturing the clones that produceantibodies to the antigen, and isolating the antibodies from thehybridoma cultures.

MAbs can be isolated and purified from hybridoma cultures by a varietyof well-established techniques. Such isolation techniques includeaffinity chromatography with Protein-A Sepharose, size-exclusionchromatography, and ion-exchange chromatography. See, for example,Coligan at pages 2.7.1-23.12 and pages 2.9.1-2.9.3. Also, see Baines etal., “Purification of Immunoglobulin G (IgG),” in METHODS IN MOLECULARBIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

After the initial raising of antibodies to the immunogen, the antibodiescan be sequenced and subsequently prepared by recombinant techniques.Humanization and chimerization of murine antibodies and antibodyfragments are well known to those skilled in the art. The use ofantibody components derived from humanized, chimeric or human antibodiesobviates potential problems associated with the immunogenicity of murineconstant regions.

Chimeric Antibodies

A chimeric antibody is a recombinant protein in which the variableregions of a human antibody have been replaced by the variable regionsof, for example, a mouse antibody, including thecomplementarity-determining regions (CDRs) of the mouse antibody.Chimeric antibodies exhibit decreased immunogenicity and increasedstability when administered to a subject. General techniques for cloningmurine immunoglobulin variable domains are disclosed, for example, inOrlandi et al., Proc. Nat'l Acad. Sci. USA 86: 3833 (1989). Techniquesfor constructing chimeric antibodies are well known to those of skill inthe art. As an example, Leung et al., Hybridoma 13:469 (1994), producedan LL2 chimera by combining DNA sequences encoding the V_(κ) and V_(H)domains of murine LL2, an anti-CD22 monoclonal antibody, with respectivehuman κ and IgG₁ constant region domains.

Humanized Antibodies

Techniques for producing humanized MAbs are well known in the art (see,e.g., Jones et al., Nature 321: 522 (1986), Riechmann et al., Nature332: 323 (1988), Verhoeyen et al., Science 239: 1534 (1988), Carter etal., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992), Sandhu, Crit. Rev.Biotech. 12: 437 (1992), and Singer et al., J. Immun. 150: 2844 (1993)).A chimeric or murine monoclonal antibody may be humanized bytransferring the mouse CDRs from the heavy and light variable chains ofthe mouse immunoglobulin into the corresponding variable domains of ahuman antibody. The mouse framework regions (FR) in the chimericmonoclonal antibody are also replaced with human FR sequences. As simplytransferring mouse CDRs into human FRs often results in a reduction oreven loss of antibody affinity, additional modification might berequired in order to restore the original affinity of the murineantibody. This can be accomplished by the replacement of one or morehuman residues in the FR regions with their murine counterparts toobtain an antibody that possesses good binding affinity to its epitope.See, for example, Tempest et al., Biotechnology 9:266 (1991) andVerhoeyen et al., Science 239: 1534 (1988). Generally, those human FRamino acid residues that differ from their murine counterparts and arelocated close to or touching one or more CDR amino acid residues wouldbe candidates for substitution.

Human Antibodies

Methods for producing fully human antibodies using either combinatorialapproaches or transgenic animals transformed with human immunoglobulinloci are known in the art (e.g., Mancini et al., 2004, New Microbiol.27:315-28; Conrad and Scheller, 2005, Comb. Chem. High ThroughputScreen. 8:117-26; Brekke and Loset, 2003, Curr. Opin. Phamacol.3:544-50). A fully human antibody also can be constructed by genetic orchromosomal transfection methods, as well as phage display technology,all of which are known in the art. See for example, McCafferty et al.,Nature 348:552-553 (1990). Such fully human antibodies are expected toexhibit even fewer side effects than chimeric or humanized antibodiesand to function in vivo as essentially endogenous human antibodies. Incertain embodiments, the claimed methods and procedures may utilizehuman antibodies produced by such techniques.

In one alternative, the phage display technique may be used to generatehuman antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res.4:126-40). Human antibodies may be generated from normal humans or fromhumans that exhibit a particular disease state, such as cancer(Dantas-Barbosa et al., 2005). The advantage to constructing humanantibodies from a diseased individual is that the circulating antibodyrepertoire may be biased towards antibodies against disease-associatedantigens.

In one non-limiting example of this methodology, Dantas-Barbosa et al.(2005) constructed a phage display library of human Fab antibodyfragments from osteosarcoma patients. Generally, total RNA was obtainedfrom circulating blood lymphocytes (Id.). Recombinant Fab were clonedfrom the μ, γ and κ chain antibody repertoires and inserted into a phagedisplay library (Id.). RNAs were converted to cDNAs and used to make FabcDNA libraries using specific primers against the heavy and light chainimmunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97).Library construction was performed according to Andris-Widhopf et al.(2000, In: Phage Display Laboratory Manual, Barbas et al. (eds), 1^(st)edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.pp. 9.1 to 9.22). The final Fab fragments were digested with restrictionendonucleases and inserted into the bacteriophage genome to make thephage display library. Such libraries may be screened by standard phagedisplay methods, as known in the art (see, e.g., Pasqualini andRuoslahti, 1996, Nature 380:364-366; Pasqualini, 1999, The Quart. J.Nucl. Med. 43:159-162).

Phage display can be performed in a variety of formats, for theirreview, see e.g. Johnson and Chiswell, Current Opinion in StructuralBiology 3:5564-571 (1993). Human antibodies may also be generated by invitro activated B-cells. See U.S. Pat. Nos. 5,567,610 and 5,229,275,incorporated herein by reference in their entirety. The skilled artisanwill realize that these techniques are exemplary and any known methodfor making and screening human antibodies or antibody fragments may beutilized.

In another alternative, transgenic animals that have been geneticallyengineered to produce human antibodies may be used to generateantibodies against essentially any immunogenic target, using standardimmunization protocols. Methods for obtaining human antibodies fromtransgenic mice are disclosed by Green et al., Nature Genet. 7:13(1994), Lonberg et al., Nature 368:856 (1994), and Taylor et al., Int.Immun. 6:579 (1994). A non-limiting example of such a system is theXenoMouse® (e.g., Green et al., 1999, J. Immunol. Methods 231:11-23)from Abgenix (Fremont, Calif.). In the XenoMouse® and similar animals,the mouse antibody genes have been inactivated and replaced byfunctional human antibody genes, while the remainder of the mouse immunesystem remains intact.

The XenoMouse® was transformed with germline-configured YACs (yeastartificial chromosomes) that contained portions of the human IgH andIgkappa loci, including the majority of the variable region sequences,along accessory genes and regulatory sequences. The human variableregion repertoire may be used to generate antibody producing B-cells,which may be processed into hybridomas by known techniques. A XenoMouse®immunized with a target antigen will produce human antibodies by thenormal immune response, which may be harvested and/or produced bystandard techniques discussed above. A variety of strains of XenoMouse®are available, each of which is capable of producing a different classof antibody. Transgenically produced human antibodies have been shown tohave therapeutic potential, while retaining the pharmacokineticproperties of normal human antibodies (Green et al., 1999). The skilledartisan will realize that the claimed compositions and methods are notlimited to use of the XenoMouse® system but may utilize any transgenicanimal that has been genetically engineered to produce human antibodies.

Antibody Fragments

Antibody fragments which recognize specific epitopes can be generated byknown techniques. Antibody fragments are antigen binding portions of anantibody, such as F(ab′)₂, Fab′, F(ab)₂, Fab, Fv, sFv and the like.F(ab′)₂ fragments can be produced by pepsin digestion of the antibodymolecule and Fab′ fragments can be generated by reducing disulfidebridges of the F(ab′)₂ fragments. Alternatively, Fab′ expressionlibraries can be constructed (Huse et al., 1989, Science, 246:1274-1281)to allow rapid and easy identification of monoclonal Fab′ fragments withthe desired specificity. F(ab)₂ fragments may be generated by papaindigestion of an antibody and Fab fragments obtained by disulfidereduction.

A single chain Fv molecule (scFv) comprises a VL domain and a VH domain.The VL and VH domains associate to form a target binding site. These twodomains are further covalently linked by a peptide linker (L). Methodsfor making scFv molecules and designing suitable peptide linkers aredescribed in U.S. Pat. No. 4,704,692, U.S. Pat. No. 4,946,778, R. Raagand M. Whitlow, “Single Chain Fvs.” FASEB Vol 9:73-80 (1995) and R. E.Bird and B. W. Walker, “Single Chain Antibody Variable Regions,”TIBTECH, Vol 9: 132-137 (1991).

Techniques for producing single domain antibodies (DABs) are also knownin the art, as disclosed for example in Cossins et al. (2006, ProtExpress Purif 51:253-259), incorporated herein by reference.

An antibody fragment can be prepared by proteolytic hydrolysis of thefull length antibody or by expression in E. coli or another host of theDNA coding for the fragment. An antibody fragment can be obtained bypepsin or papain digestion of full length antibodies by conventionalmethods. These methods are described, for example, by Goldenberg, U.S.Pat. Nos. 4,036,945 and 4,331,647 and references contained therein.Also, see Nisonoff et al., Arch Biochem. Biophys. 89: 230 (1960);Porter, Biochem. J. 73: 119 (1959), Edelman et al., in METHODS INENZYMOLOGY VOL. 1, page 422 (Academic Press 1967), and Coligan at pages2.8.1-2.8.10 and 2.10.-2.10.4.

Known Antibodies

Antibodies of use may be commercially obtained from a wide variety ofknown sources. For example, a variety of antibody secreting hybridomalines are available from the American Type Culture Collection (ATCC,Manassas, Va.). A large number of antibodies against various diseasetargets, including but not limited to tumor-associated antigens, havebeen deposited at the ATCC and/or have published variable regionsequences and are available for use in the claimed methods andcompositions. See, e.g., U.S. Pat. Nos. 7,312,318; 7,282,567; 7,151,164;7,074,403; 7,060,802; 7,056,509; 7,049,060; 7,045,132; 7,041,803;7,041,802; 7,041,293; 7,038,018; 7,037,498; 7,012,133; 7,001,598;6,998,468; 6,994,976; 6,994,852; 6,989,241; 6,974,863; 6,965,018;6,964,854; 6,962,981; 6,962,813; 6,956,107; 6,951,924; 6,949,244;6,946,129; 6,943,020; 6,939,547; 6,921,645; 6,921,645; 6,921,533;6,919,433; 6,919,078; 6,916,475; 6,905,681; 6,899,879; 6,893,625;6,887,468; 6,887,466; 6,884,594; 6,881,405; 6,878,812; 6,875,580;6,872,568; 6,867,006; 6,864,062; 6,861,511; 6,861,227; 6,861,226;6,838,282; 6,835,549; 6,835,370; 6,824,780; 6,824,778; 6,812,206;6,793,924; 6,783,758; 6,770,450; 6,767,711; 6,764,688; 6,764,681;6,764,679; 6,743,898; 6,733,981; 6,730,307; 6,720,155; 6,716,966;6,709,653; 6,693,176; 6,692,908; 6,689,607; 6,689,362; 6,689,355;6,682,737; 6,682,736; 6,682,734; 6,673,344; 6,653,104; 6,652,852;6,635,482; 6,630,144; 6,610,833; 6,610,294; 6,605,441; 6,605,279;6,596,852; 6,592,868; 6,576,745; 6,572,856; 6,566,076; 6,562,618;6,545,130; 6,544,749; 6,534,058; 6,528,625; 6,528,269; 6,521,227;6,518,404; 6,511,665; 6,491,915; 6,488,930; 6,482,598; 6,482,408;6,479,247; 6,468,531; 6,468,529; 6,465,173; 6,461,823; 6,458,356;6,455,044; 6,455,040, 6,451,310; 6,444,206′6,441,143; 6,432,404;6,432,402; 6,419,928; 6,413,726; 6,406,694; 6,403,770; 6,403,091;6,395,276; 6,395,274; 6,387,350; 6,383,759; 6,383,484; 6,376,654;6,372,215; 6,359,126; 6,355,481; 6,355,444; 6,355,245; 6,355,244;6,346,246; 6,344,198; 6,340,571; 6,340,459; 6,331,175; 6,306,393;6,254,868; 6,187,287; 6,183,744; 6,129,914; 6,120,767; 6,096,289;6,077,499; 5,922,302; 5,874,540; 5,814,440; 5,798,229; 5,789,554;5,776,456; 5,736,119; 5,716,595; 5,677,136; 5,587,459; 5,443,953,5,525,338. These are exemplary only and a wide variety of otherantibodies and their hybridomas are known in the art. The skilledartisan will realize that antibody sequences or antibody-secretinghybridomas against almost any disease-associated antigen may be obtainedby a simple search of the ATCC, NCBI and/or USPTO databases forantibodies against a selected disease-associated target of interest. Theantigen binding domains of the cloned antibodies may be amplified,excised, ligated into an expression vector, transfected into an adaptedhost cell and used for protein production, using standard techniqueswell known in the art.

Amino Acid Substitutions

In certain embodiments, the disclosed methods and compositions mayinvolve production and use of proteins or peptides with one or moresubstituted amino acid residues. As discussed above, methods for makingmonoclonal antibodies against virtually any target antigen are wellknown in the art. Typically, these result in production of murineantibodies against a target antigen. As is well known in the art, theantigen-binding specificity of murine monoclonal antibodies isdetermined largely by the hypervariable complementarity determiningregion (CDR) sequences. Murine antibodies generally comprise 6 CDRsequences, 3 on the antibody light chain and 3 on the heavy chain. Asdescribed in detail above, chimeric, humanized or human versions ofmurine antibodies may be constructed by techniques such as CDR grafting,where the murine CDR sequences are inserted into, for example, humanantibody framework and constant region sequences, or by attaching theentire murine variable region sequences to human antibody constantregion sequences. In alternative embodiments, the variable regionsequences of an antibody may be constructed, for example, by chemicalsynthesis and assembly of oligonucleotides encoding the entire light andheavy chain variable regions of an antibody.

In various embodiments, the structural, physical and/or therapeuticcharacteristics of native, chimeric, humanized or human antibodies,cytokines, or AD or DDD sequences may be optimized by replacing one ormore amino acid residues. For example, it is well known in the art thatthe functional characteristics of humanized antibodies may be improvedby substituting a limited number of human framework region (FR) aminoacids with the corresponding FR amino acids of the parent murineantibody. This is particularly true when the framework region amino acidresidues are in close proximity to the CDR residues.

In other cases, the therapeutic properties of an antibody, such asbinding affinity for the target antigen, the dissociation- or off-rateof the antibody from its target antigen, or even the effectiveness ofinduction of CDC (complement-dependent cytotoxicity) or ADCC (antibodydependent cellular cytotoxicity) by the antibody, may be optimized by alimited number of amino acid substitutions.

In alternative embodiments, the DDD and/or AD sequences used to make thecytokine-MAb DNL constructs may be further optimized, for example toincrease the DDD-AD binding affinity. Potential sequence variations inDDD or AD sequences are discussed above.

The skilled artisan will be aware that, in general, amino acidsubstitutions typically involve the replacement of an amino acid withanother amino acid of relatively similar properties (i.e., conservativeamino acid substitutions). The properties of the various amino acids andeffect of amino acid substitution on protein structure and function havebeen the subject of extensive study and knowledge in the art.

For example, the hydropathic index of amino acids may be considered(Kyte & Doolittle, 1982, J. Mol. Biol., 157:105-132). The relativehydropathic character of the amino acid contributes to the secondarystructure of the resultant protein, which in turn defines theinteraction of the protein with other molecules. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics (Kyte & Doolittle, 1982), these are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5). In making conservative substitutions, the use of amino acidswhose hydropathic indices are within ±2 is preferred, within ±1 are morepreferred, and within ±0.5 are even more preferred.

Amino acid substitution may also take into account the hydrophilicity ofthe amino acid residue (e.g., U.S. Pat. No. 4,554,101). Hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5.+-0.1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). Replacement ofamino acids with others of similar hydrophilicity is preferred.

Other considerations include the size of the amino acid side chain. Forexample, it would generally not be preferred to replace an amino acidwith a compact side chain, such as glycine or serine, with an amino acidwith a bulky side chain, e.g., tryptophan or tyrosine. The effect ofvarious amino acid residues on protein secondary structure is also aconsideration. Through empirical study, the effect of different aminoacid residues on the tendency of protein domains to adopt analpha-helical, beta-sheet or reverse turn secondary structure has beendetermined and is known in the art (see, e.g., Chou & Fasman, 1974,Biochemistry, 13:222-245; 1978, Ann. Rev. Biochem., 47: 251-276; 1979,Biophys. J., 26:367-384).

Based on such considerations and extensive empirical study, tables ofconservative amino acid substitutions have been constructed and areknown in the art. For example: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine. Alternatively: Ala (A) leu, ile, val; Arg (R)gln, asn, lys; Asn (N) his, asp, lys, arg, gln; Asp (D) asn, glu; Cys(C) ala, ser; Gln (Q) glu, asn; Glu (E) gln, asp; Gly (G) ala; His (H)asn, gln, lys, arg; Ile (I) val, met, ala, phe, leu; Leu (L) val, met,ala, phe, ile; Lys (K) gln, asn, arg; Met (M) phe, ile, leu; Phe (F)leu, val, ile, ala, tyr; Pro (P) ala; Ser (S), thr; Thr (T) ser; Trp (W)phe, tyr; Tyr (Y) trp, phe, thr, ser; Val (V) ile, leu, met, phe, ala.

Other considerations for amino acid substitutions include whether or notthe residue is located in the interior of a protein or is solventexposed. For interior residues, conservative substitutions wouldinclude: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala andGly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr;Tyr and Trp. (See, e.g., PROWL website at rockefeller.edu) For solventexposed residues, conservative substitutions would include: Asp and Asn;Asp and Glu; Glu and Gln; Glu and Ala; Gly and Asn; Ala and Pro; Ala andGly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu;Leu and Ile; Ile and Val; Phe and Tyr. (Id.) Various matrices have beenconstructed to assist in selection of amino acid substitutions, such asthe PAM250 scoring matrix, Dayhoff matrix, Grantham matrix, McLachlanmatrix, Doolittle matrix, Henikoff matrix, Miyata matrix, Fitch matrix,Jones matrix, Rao matrix, Levin matrix and Risler matrix (Idem.)

In determining amino acid substitutions, one may also consider theexistence of intermolecular or intramolecular bonds, such as formationof ionic bonds (salt bridges) between positively charged residues (e.g.,His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) ordisulfide bonds between nearby cysteine residues.

Methods of substituting any amino acid for any other amino acid in anencoded protein sequence are well known and a matter of routineexperimentation for the skilled artisan, for example by the technique ofsite-directed mutagenesis or by synthesis and assembly ofoligonucleotides encoding an amino acid substitution and splicing intoan expression vector construct.

Therapeutic Agents

In alternative embodiments, therapeutic agents such as cytotoxic agents,anti-angiogenic agents, pro-apoptotic agents, antibiotics, hormones,hormone antagonists, chemokines, drugs, prodrugs, toxins, enzymes orother agents may be used as adjunct therapies to the multimericcytokines described herein. Drugs of use may possess a pharmaceuticalproperty selected from the group consisting of antimitotic, antikinase,alkylating, antimetabolite, antibiotic, alkaloid, anti-angiogenic,pro-apoptotic agents and combinations thereof.

Exemplary drugs of use may include 5-fluorouracil, aplidin, azaribine,anastrozole, anthracyclines, bendamustine, bleomycin, bortezomib,bryostatin-1, busulfan, calicheamycin, camptothecin, carboplatin,10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin(CDDP), Cox-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin,cladribine, camptothecans, cyclophosphamide, cytarabine, dacarbazine,docetaxel, dactinomycin, daunorubicin, doxorubicin,2-pyrrolinodoxorubicine (2P-DOX), cyano-morpholino doxorubicin,doxorubicin glucuronide, epirubicin glucuronide, estramustine,epipodophyllotoxin, estrogen receptor binding agents, etoposide (VP16),etoposide glucuronide, etoposide phosphate, floxuridine (FUdR),3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide,farnesyl-protein transferase inhibitors, gemcitabine, hydroxyurea,idarubicin, ifosfamide, L-asparaginase, lenolidamide, leucovorin,lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine,methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine,nitrosurea, plicomycin, procarbazine, paclitaxel, pentostatin, PSI-341,raloxifene, semustine, streptozocin, tamoxifen, taxol, temazolomide (anaqueous form of DTIC), transplatinum, thalidomide, thioguanine,thiotepa, teniposide, topotecan, uracil mustard, vinorelbine,vinblastine, vincristine and vinca alkaloids.

Toxins of use may include ricin, abrin, alpha toxin, saporin,ribonuclease (RNase), e.g., onconase, DNase I, Staphylococcalenterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin,Pseudomonas exotoxin, and Pseudomonas endotoxin.

Chemokines of use may include RANTES, MCAF, MIP1-alpha, MIP1-Beta andIP-10.

In certain embodiments, anti-angiogenic agents, such as angiostatin,baculostatin, canstatin, maspin, anti-VEGF antibodies, anti-P1GFpeptides and antibodies, anti-vascular growth factor antibodies,anti-Flk-1 antibodies, anti-Flt-1 antibodies and peptides, anti-Krasantibodies, anti-cMET antibodies, anti-MIF (macrophagemigration-inhibitory factor) antibodies, laminin peptides, fibronectinpeptides, plasminogen activator inhibitors, tissue metalloproteinaseinhibitors, interferons, interleukin-12, IP-10, Gro-β, thrombospondin,2-methoxyoestradiol, proliferin-related protein, carboxiamidotriazole,CM101, Marimastat, pentosan polysulphate, angiopoietin-2,interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment,Linomide (roquinimex), thalidomide, pentoxifylline, genistein, TNP-470,endostatin, paclitaxel, accutin, angiostatin, cidofovir, vincristine,bleomycin, AGM-1470, platelet factor 4 or minocycline may be of use.

Other useful therapeutic agents may comprise oligonucleotides,especially antisense oligonucleotides that preferably are directedagainst oncogenes and oncogene products, such as bcl-2 or p53. Apreferred form of therapeutic oligonucleotide is siRNA.

Diagnostic Agents

Diagnostic agents are preferably selected from the group consisting of aradionuclide, a radiological contrast agent, a paramagnetic ion, ametal, a fluorescent label, a chemiluminescent label, an ultrasoundcontrast agent and a photoactive agent. Such diagnostic agents are wellknown and any such known diagnostic agent may be used. Non-limitingexamples of diagnostic agents may include a radionuclide such as ¹¹⁰In,¹¹¹In, ¹⁷⁷Lu, ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁹⁰Y, ⁸⁹Zr,^(94m)Tc, ⁹⁴Tc, ^(99m)Tc, ¹²⁰I, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁵⁴⁻¹⁵⁸Gd, ³²P,¹¹C, ¹³N, ¹⁵O, ¹⁸⁶Re, ¹⁸⁸Re, ⁵¹Mn, ^(52m)Mn, ⁵⁵Co, ⁷²As, ⁷⁵Br, ⁷⁶Br,^(82m)Rb, ⁸³Sr, or other gamma-, beta-, or positron-emitters.Paramagnetic ions of use may include chromium (III), manganese (II),iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium(III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II),terbium (III), dysprosium (III), holmium (III) or erbium (III). Metalcontrast agents may include lanthanum (III), gold (III), lead (II) orbismuth (III). Ultrasound contrast agents may comprise liposomes, suchas gas filled liposomes. Radiopaque diagnostic agents may be selectedfrom compounds, barium compounds, gallium compounds, and thalliumcompounds. A wide variety of fluorescent labels are known in the art,including but not limited to fluorescein isothiocyanate, rhodamine,phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde andfluorescamine. Chemiluminescent labels of use may include luminol,isoluminol, an aromatic acridinium ester, an imidazole, an acridiniumsalt or an oxalate ester.

Immunoconjugates

In certain embodiments, the antibody component of the cytokine-MAb DNLconstruct may be conjugated to one or more therapeutic or diagnosticagents. The therapeutic agents do not need to be the same but can bedifferent, e.g. a drug and a radioisotope. For example, ¹³¹I can beincorporated into a tyrosine of an antibody or fusion protein and a drugattached to an epsilon amino group of a lysine residue. Therapeutic anddiagnostic agents also can be attached, for example to reduced SH groupsand/or to carbohydrate side chains. Many methods for making covalent ornon-covalent conjugates of therapeutic or diagnostic agents withantibodies or fusion proteins are known in the art and any such knownmethod may be utilized.

A therapeutic or diagnostic agent can be attached at the hinge region ofa reduced antibody component via disulfide bond formation.Alternatively, such agents can be attached using a heterobifunctionalcross-linker, such as N-succinyl 3-(2-pyridyldithio)propionate (SPDP).Yu et al., Int. J. Cancer 56: 244 (1994). General techniques for suchconjugation are well-known in the art. See, for example, Wong, CHEMISTRYOF PROTEIN CONJUGATION AND CROSS-LINKING (CRC Press 1991); Upeslacis etal., “Modification of Antibodies by Chemical Methods,” in MONOCLONALANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al. (eds.), pages187-230 (Wiley-Liss, Inc. 1995); Price, “Production and Characterizationof Synthetic Peptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES:PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.),pages 60-84 (Cambridge University Press 1995). Alternatively, thetherapeutic or diagnostic agent can be conjugated via a carbohydratemoiety in the Fc region of the antibody. The carbohydrate group can beused to increase the loading of the same agent that is bound to a thiolgroup, or the carbohydrate moiety can be used to bind a differenttherapeutic or diagnostic agent.

Methods for conjugating peptides to antibody components via an antibodycarbohydrate moiety are well-known to those of skill in the art. See,for example, Shih et al., Int. J. Cancer 41: 832 (1988); Shih et al.,Int. J. Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No.5,057,313, incorporated herein in their entirety by reference. Thegeneral method involves reacting an antibody component having anoxidized carbohydrate portion with a carrier polymer that has at leastone free amine function. This reaction results in an initial Schiff base(imine) linkage, which can be stabilized by reduction to a secondaryamine to form the final conjugate.

The Fc region may be absent if the antibody used as the antibodycomponent of the immunoconjugate is an antibody fragment. However, it ispossible to introduce a carbohydrate moiety into the light chainvariable region of a full length antibody or antibody fragment. See, forexample, Leung et al., J. Immunol. 154: 5919 (1995); Hansen et al., U.S.Pat. No. 5,443,953 (1995), Leung et al., U.S. Pat. No. 6,254,868,incorporated herein by reference in their entirety. The engineeredcarbohydrate moiety is used to attach the therapeutic or diagnosticagent.

In some embodiments, a chelating agent may be attached to an antibody,antibody fragment or fusion protein and used to chelate a therapeutic ordiagnostic agent, such as a radionuclide. Exemplary chelators includebut are not limited to DTPA (such as Mx-DTPA), DOTA, TETA, NETA or NOTA.Methods of conjugation and use of chelating agents to attach metals orother ligands to proteins are well known in the art (see, e.g., U.S.patent application Ser. No. 12/112,289, incorporated herein by referencein its entirety).

In certain embodiments, radioactive metals or paramagnetic ions may beattached to proteins or peptides by reaction with a reagent having along tail, to which may be attached a multiplicity of chelating groupsfor binding ions. Such a tail can be a polymer such as a polylysine,polysaccharide, or other derivatized or derivatizable chains havingpendant groups to which can be bound chelating groups such as, e.g.,ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DIVA), porphyrins, polyamines, crown ethers,bis-thiosemicarbazones, polyoximes, and like groups known to be usefulfor this purpose.

Chelates may be directly linked to antibodies or peptides, for exampleas disclosed in U.S. Pat. No. 4,824,659, incorporated herein in itsentirety by reference. Particularly useful metal-chelate combinationsinclude 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, usedwith diagnostic isotopes in the general energy range of 60 to 4,000 keV,such as ¹²⁵I, ¹³¹I, ¹²³I, ¹²⁴I, ⁶²Cu, ⁶⁴Cu, ¹⁸F, ¹¹¹In, ⁶⁷Ga, ⁶⁸Ga,^(99m)TC, ^(94m)TC, ¹¹C, ¹³N, ¹⁵O, ⁷⁶Br, for radioimaging. The samechelates, when complexed with non-radioactive metals, such as manganese,iron and gadolinium are useful for MRI. Macrocyclic chelates such asNOTA, DOTA, and TETA are of use with a variety of metals andradiometals, most particularly with radionuclides of gallium, yttriumand copper, respectively. Such metal-chelate complexes can be made verystable by tailoring the ring size to the metal of interest. Otherring-type chelates such as macrocyclic polyethers, which are of interestfor stably binding nuclides, such as ²²³Ra for RAIT are encompassed.

More recently, methods of ¹⁸F-labeling of use in PET scanning techniqueshave been disclosed, for example by reaction of F-18 with a metal orother atom, such as aluminum. The ¹⁸F-Al conjugate may be complexed withchelating groups, such as DOTA, NOTA or NETA that are attached directlyto antibodies or used to label targetable constructs in pre-targetingmethods. Such F-18 labeling techniques are disclosed in U.S. patentapplication Ser. No. 12/112,289, filed Apr. 30, 2008, the entire text ofwhich is incorporated herein by reference.

Methods of Therapeutic Treatment

Various embodiments concern methods of treating a cancer in a subject,such as a mammal, including humans, domestic or companion pets, such asdogs and cats, comprising administering to the subject a therapeuticallyeffective amount of a cytokine-MAb DNL construct. In preferredembodiments, the cytokine-MAb DNL construct is a 20-2b construct, asdescribed in further detail in the Examples below.

The administration of cytokine-MAb DNL construct can be supplemented byadministering concurrently or sequentially a therapeutically effectiveamount of another antibody that binds to or is reactive with anotherantigen on the surface of the target cell. Preferred additional MAbscomprise at least one humanized, chimeric or human MAb selected from thegroup consisting of a MAb reactive with CD4, CD5, CD8, CD14, CD15, CD16,CD19, IGF-1R, CD20, CD21, CD22, CD23, CD25, CD30, CD32b, CD33, CD37,CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD70, CD74, CD79a, CD80,CD95, CD126, CD133, CD138, CD154, CEACAM5, CEACAM6, B7, AFP, PSMA,EGP-1, EGP-2, carbonic anhydrase IX, PAM4 antigen, MUC1, MUC2, MUC3,MUC4, MUC5, Ia, MIF, HM1.24, HLA-DR, tenascin, Flt-3, VEGFR, P1GF, ILGF,IL-6, IL-25, tenascin, TRAIL-R1, TRAIL-R2, complement factor C5,oncogene product, or a combination thereof. Various antibodies of use,such as anti-CD19, anti-CD20, and anti-CD22 antibodies, are known tothose of skill in the art. See, for example, Ghetie et al., Cancer Res.48:2610 (1988); Hekman et al., Cancer Immunol. Immunother. 32:364(1991); Longo, Curr. Opin. Oncol. 8:353 (1996), U.S. Pat. Nos.5,798,554; 6,187,287; 6,306,393; 6,676,924; 7,109,304; 7,151,164;7,230,084; 7,230,085; 7,238,785; 7,238,786; 7,282,567; 7,300,655;7,312,318; and U.S. Patent Application Publ. Nos. 20080131363;20080089838; 20070172920; 20060193865; 20060210475; 20080138333; and20080146784, each incorporated herein by reference.

The cytokine-MAb DNL construct therapy can be further supplemented withthe administration, either concurrently or sequentially, of at least onetherapeutic agent. For example, “CVB” (1.5 g/m² cyclophosphamide,200-400 mg/m² etoposide, and 150-200 mg/m² carmustine) is a regimen usedto treat non-Hodgkin's lymphoma. Patti et al., Eur. J. Haematol. 51: 18(1993). Other suitable combination chemotherapeutic regimens arewell-known to those of skill in the art. See, for example, Freedman etal., “Non-Hodgkin's Lymphomas,” in CANCER MEDICINE, VOLUME 2, 3rdEdition, Holland et al. (eds.), pages 2028-2068 (Lea & Febiger 1993). Asan illustration, first generation chemotherapeutic regimens fortreatment of intermediate-grade non-Hodgkin's lymphoma (NHL) includeC-MOPP (cyclophosphamide, vincristine, procarbazine and prednisone) andCHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone). Auseful second generation chemotherapeutic regimen is m-BACOD(methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine,dexamethasone and leucovorin), while a suitable third generation regimenis MACOP-B (methotrexate, doxorubicin, cyclophosphamide, vincristine,prednisone, bleomycin and leucovorin). Additional useful drugs includephenyl butyrate, bendamustine, and bryostatin-1.

The cytokine-MAb DNL construct can be formulated according to knownmethods to prepare pharmaceutically useful compositions, whereby thecytokine-MAb DNL construct is combined in a mixture with apharmaceutically suitable excipient. Sterile phosphate-buffered salineis one example of a pharmaceutically suitable excipient. Other suitableexcipients are well-known to those in the art. See, for example, Anselet al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5thEdition (Lea & Febiger 1990), and Gennaro (ed.), REMINGTON′SPHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Company 1990),and revised editions thereof.

The cytokine-MAb DNL construct can be formulated for intravenousadministration via, for example, bolus injection or continuous infusion.Preferably, cytokine-MAb DNL construct is infused over a period of lessthan about 4 hours, and more preferably, over a period of less thanabout 3 hours. For example, the first 25-50 mg could be infused within30 minutes, preferably even 15 min, and the remainder infused over thenext 2-3 hrs. Formulations for injection can be presented in unit dosageform, e.g., in ampoules or in multi-dose containers, with an addedpreservative. The compositions can take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and can containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient can be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

Additional pharmaceutical methods may be employed to control theduration of action of the cytokine-MAb DNL construct. Control releasepreparations can be prepared through the use of polymers to complex oradsorb the cytokine-MAb DNL construct. For example, biocompatiblepolymers include matrices of poly(ethylene-co-vinyl acetate) andmatrices of a polyanhydride copolymer of a stearic acid dimer andsebacic acid. Sherwood et al., Bio/Technology 10: 1446 (1992). The rateof release from such a matrix depends upon the molecular weight of thecytokine-MAb DNL construct, the amount of cytokine-MAb DNL constructwithin the matrix, and the size of dispersed particles. Saltzman et al.,Biophys. J. 55: 163 (1989); Sherwood et al., supra. Other solid dosageforms are described in Ansel et al., PHARMACEUTICAL DOSAGE FORMS ANDDRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro(ed.), REMINGTON′S PHARMACEUTICAL SCIENCES, 18th Edition (MackPublishing Company 1990), and revised editions thereof.

The cytokine-MAb DNL construct may also be administered to a mammalsubcutaneously or even by other parenteral routes. Moreover, theadministration may be by continuous infusion or by single or multipleboluses. Preferably, the cytokine-MAb DNL construct is infused over aperiod of less than about 4 hours, and more preferably, over a period ofless than about 3 hours.

More generally, the dosage of an administered cytokine-MAb DNL constructfor humans will vary depending upon such factors as the patient's age,weight, height, sex, general medical condition and previous medicalhistory. It may be desirable to provide the recipient with a dosage ofcytokine-MAb DNL construct that is in the range of from about 1 mg/kg to25 mg/kg as a single intravenous infusion, although a lower or higherdosage also may be administered as circumstances dictate. A dosage of1-20 mg/kg for a 70 kg patient, for example, is 70-1,400 mg, or 41-824mg/m² for a 1.7-m patient. The dosage may be repeated as needed, forexample, once per week for 4-10 weeks, once per week for 8 weeks, oronce per week for 4 weeks. It may also be given less frequently, such asevery other week for several months, or monthly or quarterly for manymonths, as needed in a maintenance therapy.

Alternatively, a cytokine-MAb DNL construct may be administered as onedosage every 2 or 3 weeks, repeated for a total of at least 3 dosages.Or, the construct may be administered twice per week for 4-6 weeks. Ifthe dosage is lowered to approximately 200-300 mg/m² (340 mg per dosagefor a 1.7-m patient, or 4.9 mg/kg for a 70 kg patient), it may beadministered once or even twice weekly for 4 to 10 weeks. Alternatively,the dosage schedule may be decreased, namely every 2 or 3 weeks for 2-3months. It has been determined, however, that even higher doses, such as20 mg/kg once weekly or once every 2-3 weeks can be administered by slowi.v. infusion, for repeated dosing cycles. The dosing schedule canoptionally be repeated at other intervals and dosage may be giventhrough various parenteral routes, with appropriate adjustment of thedose and schedule.

In preferred embodiments, the cytokine-MAb DNL constructs are of use fortherapy of cancer. Examples of cancers include, but are not limited to,carcinoma, lymphoma, glioblastoma, melanoma, sarcoma, and leukemia,myeloma, or lymphoid malignancies. More particular examples of suchcancers are noted below and include: squamous cell cancer (e.g.,epithelial squamous cell cancer), Ewing sarcoma, Wilms tumor,astrocytomas, lung cancer including small-cell lung cancer, non-smallcell lung cancer, adenocarcinoma of the lung and squamous carcinoma ofthe lung, cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer including gastrointestinal cancer, pancreatic cancer,glioblastoma multiforme, cervical cancer, ovarian cancer, liver cancer,bladder cancer, hepatoma, hepatocellular carcinoma, neuroendocrinetumors, medullary thyroid cancer, differentiated thyroid carcinoma,breast cancer, ovarian cancer, colon cancer, rectal cancer, endometrialcancer or uterine carcinoma, salivary gland carcinoma, kidney or renalcancer, prostate cancer, vulvar cancer, anal carcinoma, penilecarcinoma, as well as head-and-neck cancer. The term “cancer” includesprimary malignant cells or tumors (e.g., those whose cells have notmigrated to sites in the subject's body other than the site of theoriginal malignancy or tumor) and secondary malignant cells or tumors(e.g., those arising from metastasis, the migration of malignant cellsor tumor cells to secondary sites that are different from the site ofthe original tumor). Cancers conducive to treatment methods of thepresent invention involves cells which express, over-express, orabnormally express IGF-1R.

Other examples of cancers or malignancies include, but are not limitedto: Acute Childhood Lymphoblastic Leukemia, Acute LymphoblasticLeukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia,Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult(Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult AcuteMyeloid Leukemia, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia,Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult SoftTissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, AnalCancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer,Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of the RenalPelvis and Ureter, Central Nervous System (Primary) Lymphoma, CentralNervous System Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma,Cervical Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood(Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia,Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, ChildhoodCerebellar Astrocytoma, Childhood Cerebral Astrocytoma, ChildhoodExtracranial Germ Cell Tumors, Childhood Hodgkin's Disease, ChildhoodHodgkin's Lymphoma, Childhood Hypothalamic and Visual Pathway Glioma,Childhood Lymphoblastic Leukemia, Childhood Medulloblastoma, ChildhoodNon-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial PrimitiveNeuroectodermal Tumors, Childhood Primary Liver Cancer, ChildhoodRhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood VisualPathway and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, ChronicMyelogenous Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, EndocrinePancreas Islet Cell Carcinoma, Endometrial Cancer, Ependymoma,Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and RelatedTumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor,Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer,Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, GastricCancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, GermCell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia, Headand Neck Cancer, Hepatocellular Cancer, Hodgkin's Lymphoma,Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal Cancers,Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell PancreaticCancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and OralCavity Cancer, Liver Cancer, Lung Cancer, Lymphoproliferative Disorders,Macroglobulinemia, Male Breast Cancer, Malignant Mesothelioma, MalignantThymoma, Medulloblastoma, Melanoma, Mesothelioma, Metastatic OccultPrimary Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer,Metastatic Squamous Neck Cancer, Multiple Myeloma, MultipleMyeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, MyelogenousLeukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavityand Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma,Non-Hodgkin's Lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell LungCancer, Occult Primary Metastatic Squamous Neck Cancer, OropharyngealCancer, Osteo-/Malignant Fibrous Sarcoma, Osteosarcoma/Malignant FibrousHistiocytoma, Osteosarcoma/Malignant Fibrous Histiocytoma of Bone,Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian LowMalignant Potential Tumor, Pancreatic Cancer, Paraproteinemias,Polycythemia vera, Parathyroid Cancer, Penile Cancer, Pheochromocytoma,Pituitary Tumor, Primary Central Nervous System Lymphoma, Primary LiverCancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvisand Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary GlandCancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small CellLung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous NeckCancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal andPineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, ThyroidCancer, Transitional Cell Cancer of the Renal Pelvis and Ureter,Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors,Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer,Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma,Vulvar Cancer, Waldenstrom's Macroglobulinemia, Wilms' Tumor, and anyother hyperproliferative disease, besides neoplasia, located in an organsystem listed above.

The methods and compositions described and claimed herein may be used totreat malignant or premalignant conditions and to prevent progression toa neoplastic or malignant state, including but not limited to thosedisorders described above. Such uses are indicated in conditions knownor suspected of preceding progression to neoplasia or cancer, inparticular, where non-neoplastic cell growth consisting of hyperplasia,metaplasia, or most particularly, dysplasia has occurred (for review ofsuch abnormal growth conditions, see Robbins and Angell, BasicPathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79 (1976)).

Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia. It is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation. Dysplasticdisorders which can be treated include, but are not limited to,anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiatingthoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia,cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia,cleidocranial dysplasia, congenital ectodermal dysplasia,craniodiaphysial dysplasia, craniocarpotarsal dysplasia,craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia,ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia,dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex,dysplasia epiphysialis punctata, epithelial dysplasia,faciodigitogenital dysplasia, familial fibrous dysplasia of jaws,familial white folded dysplasia, fibromuscular dysplasia, fibrousdysplasia of bone, florid osseous dysplasia, hereditary renal-retinaldysplasia, hidrotic ectodermal dysplasia, hypohidrotic ectodermaldysplasia, lymphopenic thymic dysplasia, mammary dysplasia,mandibulofacial dysplasia, metaphysial dysplasia, Mondini dysplasia,monostotic fibrous dysplasia, mucoepithelial dysplasia, multipleepiphysial dysplasia, oculoauriculovertebral dysplasia,oculodentodigital dysplasia, oculovertebral dysplasia, odontogenicdysplasia, opthalmomandibulomelic dysplasia, periapical cementaldysplasia, polyostotic fibrous dysplasia, pseudoachondroplasticspondyloepiphysial dysplasia, retinal dysplasia, septo-optic dysplasia,spondyloepiphysial dysplasia, and ventriculoradial dysplasia.

Additional pre-neoplastic disorders which can be treated include, butare not limited to, benign dysproliferative disorders (e.g., benigntumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps oradenomas, and esophageal dysplasia), leukoplakia, keratoses, Bowen'sdisease, Farmer's Skin, solar cheilitis, and solar keratosis.

In preferred embodiments, the method of the invention is used to inhibitgrowth, progression, and/or metastasis of cancers, in particular thoselisted above.

Additional hyperproliferative diseases, disorders, and/or conditionsinclude, but are not limited to, progression, and/or metastases ofmalignancies and related disorders such as leukemia (including acuteleukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia(including myeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia)) and chronic leukemias (e.g., chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,emangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, and retinoblastoma.

Kits

Various embodiments may concern kits containing components suitable fortreating or diagnosing diseased tissue in a patient. Exemplary kits maycontain at least one or more cytokine-MAb constructs as describedherein. If the composition containing components for administration isnot formulated for delivery via the alimentary canal, such as by oraldelivery, a device capable of delivering the kit components through someother route may be included. One type of device, for applications suchas parenteral delivery, is a syringe that is used to inject thecomposition into the body of a subject. Inhalation devices may also beused. In certain embodiments, a therapeutic agent may be provided in theform of a prefilled syringe or autoinjection pen containing a sterile,liquid formulation or lyophilized preparation.

The kit components may be packaged together or separated into two ormore containers. In some embodiments, the containers may be vials thatcontain sterile, lyophilized formulations of a composition that aresuitable for reconstitution. A kit may also contain one or more bufferssuitable for reconstitution and/or dilution of other reagents. Othercontainers that may be used include, but are not limited to, a pouch,tray, box, tube, or the like. Kit components may be packaged andmaintained sterilely within the containers. Another component that canbe included is instructions to a person using a kit for its use.

Expression Vectors

Still other embodiments may concern DNA sequences comprising a nucleicacid encoding a cytokine-MAb construct, or its constituent fusionproteins. Fusion proteins may comprise an antibody or cytokine attachedto a different peptide or protein, such as the AD and DDD peptidesutilized for DNL construct formation as discussed in more detail in theExamples below.

Various embodiments relate to expression vectors comprising the codingDNA sequences. The vectors may contain sequences encoding the light andheavy chain constant regions and the hinge region of a humanimmunoglobulin to which may be attached chimeric, humanized or humanvariable region sequences. The vectors may additionally containpromoters that express the encoded protein(s) in a selected host cell,enhancers and signal or leader sequences. Vectors that are particularlyuseful are pdHL2 or GS. More preferably, the light and heavy chainconstant regions and hinge region may be from a human EU myelomaimmunoglobulin, where optionally at least one of the amino acid in theallotype positions is changed to that found in a different IgG1allotype, and wherein optionally amino acid 253 of the heavy chain of EUbased on the EU number system may be replaced with alanine. See Edelmanet al., Proc. Natl. Acad. Sci. USA 63: 78-85 (1969). In otherembodiments, an IgG1 sequence may be converted to an IgG4 sequence.

The skilled artisan will realize that methods of genetically engineeringexpression constructs and insertion into host cells to expressengineered proteins are well known in the art and a matter of routineexperimentation. Host cells and methods of expression of clonedantibodies or fragments have been described, for example, in U.S. patentapplication Ser. Nos. 11/187,863, filed Jul. 25, 2005; 11/253,666, filedOct. 20, 2005 and 11/487,215, filed Jul. 14, 2006, each incorporatedherein by reference in its entirety.

EXAMPLES

The following examples are provided to illustrate, but not to limit, theclaims of the present invention.

General Methods

Cell lines. Cell lines suitable for transfection and protein productionare known in the art, such as Sp/EEE and Sp/ESF cells (see, e.g., U.S.Pat. Nos. 7,531,327; 7,537,930 and 7,608,425, the Examples section ofeach of which is incorporated herein by reference.) Sp/ESF cells, anenhanced variant of Sp2/0-Ag14 (ATCC, Manassas, Va.), were maintained inHybridoma Serum-Free Media (H—SFM) supplemented with 2 mM L-glutamineand 100 units/mL penicillin-streptomycin. Daudi, Ramos, Raji, NAMALWAand Jeko-1 (ATCC) human lymphomas were maintained in RPMI 1640 mediacontaining 10% FBS and supplemented with 1 mM sodium pyruvate, 10 mML-glutamine, 25 mM HEPES, and 100 units/mL penicillin-streptomycin.

MAb-IFNα Constructs.

20-2b was produced by DNL via the combination of two DNL modules,C_(H3)-AD2-IgG-v-mab and IFNα2b-DDD2, which were each expressed inSp/ESF. Additional DNL-generated MAb-IFNα constructs, of similar designas 20-2b (humanized IgG1+4 IFNα2b) but with different targeting MAbs,were used as controls in several experiments: 22-2b hasC_(H3)-AD2-IgG-e-mab (epratuzumab) as its AD2 module, which is directedagainst CD22 and binds lymphoma; 734-2b has C_(H3)-AD2-IgG-h734 as itsAD2 module, which is directed against the hapten, In-DTPA and does notbind to any animal proteins or tissues; and R1-2b usesC_(H3)-AD2-IgG-hR1, which binds human insulin-like growth factor 1receptor (IGF-1R).

Analytical Methods.

Protein concentrations of IFNα constructs were measured using acommercial human IFNα2 ELISA kit following the manufacturer's suggestedprotocol (PBL Interferon Source, Piscataway, N.J.) and confirmed bysize-exclusion HPLC performed on a Beckman System Gold Model 116 with aBio-Sil SEC 250 column (Bio-Rad, Hercules, Calif.) and 0.04 M PBS, pH6.8, 1 mM EDTA as the mobile phase. Reducing and non-reducing SDS-PAGEanalyses were performed using 4-20% gradient Tris-glycine gels (CambrexBio Science, Rockland, Me.). All colorimetric (ELISA and MTS),luminescence (reporter) and fluorometric (CDC and ADCC) assays werequantified with an EnVision 2100 multilabel plate reader (PerkinElmer,Waltham, Mass.).

IFNα Activity Measurements.

IFNα2b specific activities were determined using the iLite HumanInterferon Alpha Cell-Based Assay Kit following the manufacturer'ssuggested protocol (PBL Interferon Source). PEGASYS®, 20-2b, and sevenmore MAb-IFNα constructs were diluted to 10, 2.5 and 0.625 ng/mL in 1%BSA-PBS. PEG-INTRON® was diluted to 1, 0.25 and 0.0625 ng/mL. Eachdilution was assayed in triplicate with overnight incubation with thesupplied cells. Specific activities were extrapolated from a standardcurve generated with the supplied standard. Antiviral activities weredetermined with an in vitro viral challenge assay usingencephalomyocarditis (EMC) virus on A549 cells by an independentanalytical laboratory (PBL Interferon Source).

In Vitro Proliferation.

Daudi or Jeko-1 were plated at 5,000 cells/well in 96-well plates andincubated at 37° C. for four (Daudi) or five (Jeko-1) days in thepresence of increasing concentrations of the indicated agents. Viablecell densities were determined using a CellTiter 96 Cell ProliferationAssay (Promega, Madison, Wis.).

Ex-Vivo Depletion of Daudi and Ramos Lymphoma Cells from Whole Blood.

The effects of 20-2b on NHL cells as well as peripheral bloodlymphocytes in whole human blood from health volunteers were evaluatedex-vivo using flow cytometry and compared to those of v-mab, 734-2b or acombination of v-mab and 734-2b. Daudi or Ramos (5×10⁴ cells) were mixedwith heparinized whole blood (150 μl) and incubated with test MAbs at0.01, 0.1 or 1 nM for 2 days at 37° C. and 5% CO₂. Cells were stainedwith FITC-labeled anti-CD3, anti-CD19, or mouse IgG₁ isotype control (BDBiosciences, San Jose, Calif.). Following lysis of erythrocytes, cellswere analyzed using a FACSCalibur (BD Biosciences) with Cell Questsoftware. Both Daudi and Ramos cells are CD19+ and in the monocyte gate.The normal B- and T-cells are CD19+ and CD3+ cells, respectively, in thelymphocyte gate. Student t-test was used to evaluate statisticalsignificance (P<0.05). In vivo efficacy in mice. Studies were performedin female C.B.17 homozygous severe combined immune deficient (SCID) miceof approximately 20 g (Taconic, Germantown, N.Y.). Each mouse wasinoculated i.v. with 1.5×10⁷ Daudi, 2.5×10⁶ Raji or 5×10⁶ NAMALWA cellson day 0. Treatment doses were all administered by subcutaneousinjection. Saline was used as a control treatment. Animals monitoreddaily were humanely sacrificed when hind-limb paralysis developed or ifthey became otherwise moribund. Additionally, mice were sacrificed ifthey lost more than 20% of initial body weight. Survival curves wereanalyzed using Kaplan-Meier plots (log-rank analysis), using the Prism(v4.03) software package (GraphPad Software, Inc.). Some outliersdetermined by critical Z test were censored from analyses.

Example 1 C_(H3)-AD2-IgG Expression Vectors

The pdHL2 mammalian expression vector has been used for the expressionof recombinant IgGs (Qu et al., Methods 2005, 36:84-95). A plasmidshuttle vector was produced to facilitate the conversion of anyIgG-pdHL2 vector into a C_(H3)-AD2-IgG-pdHL2 vector. The gene for the Fc(C_(H2) and C_(H3) domains) was amplified by PCR using the pdHL2 vectoras a template and the following oligonucleotide primers:

Fc BglII Left (SEQ ID NO: 21) AGATCTGGCGCACCTGAACTCCTGFc Bam- EcoRI Right (SEQ ID NO: 22) GAATTCGGATCCTTTACCCGGAGACAGGGAGAG.

The amplimer was cloned in the pGemT PCR cloning vector (Promega). TheFc insert fragment was excised from pGemT with Xba I and Bam HI andligated with AD2-pdHL2 vector that was prepared by digestingh679-Fab-AD2-pdHL2 (Rossi et al., Proc Natl Acad Sci USA 2006,103:6841-6) with Xba I and Bam HI, to generate the shuttle vectorFc-AD2-pdHL2. To convert IgG-pdHL2 expression vectors to aC_(H3)-AD2-IgG-pdHL2 expression vectors, an 861 bp BsrG I/Nde Irestriction fragment was excised from the former and replaced with a 952bp BsrG I/Nde I restriction fragment excised from the Fc-AD2-pdHL2vector. The following is a partial list of C_(H3)-AD2-IgG-pdHL2expression vectors that have been generated and used for the productionof recombinant humanized IgG-AD2 modules:

C_(H3)-AD2-IgG-hA20 (anti-CD20)

C_(H3)-AD2-IgG-hLL2 (anti-CD22)

C_(H3)-AD2-IgG-hL243 (anti-HLA-DR)

C_(H3)-AD2-IgG-hLL1 (anti-CD74)

C_(H3)-AD2-IgG-hR1 (anti-IGF-1R)

C_(H3)-AD2-IgG-h734 (anti-Indium-DTPA).

Example 2 Production of C_(H3)-AD2-IgG

Transfection and Selection of Stable C_(H3)-AD2-IgG Secreting Cell Lines

All cell lines were grown in Hybridoma SFM (Invitrogen, CarlsbadCalif.). C_(H3)-AD2-IgG-pdHL2 vectors (30 μg) were linearized bydigestion with Sal I restriction endonuclease and transfected intoSp2/0-Ag14 (2.8×10⁶ cells) by electroporation (450 volts, 25 μF). ThepdHL2 vector contains the gene for dihydrofolate reductase allowingclonal selection as well as gene amplification with methotrexate (MTX).

Following transfection, the cells were plated in 96-well plates andtransgenic clones were selected in media containing 0.2 μM MTX. Cloneswere screened for C_(H3)-AD2-IgG productivity by a sandwich ELISA using96-well microtitre plates coated with specific anti-idiotype MAbs.Conditioned media from the putative clones were transferred to themicro-plate wells and detection of the fusion protein was accomplishedwith horseradish peroxidase-conjugated goat anti-human IgG F(ab′)₂(Jackson ImmunoResearch Laboratories, West Grove, Pa.). Wells giving thehighest signal were expanded and ultimately used for production.

Production and Purification of C₁₋₃-AD2-IgG Modules

For production of the fusion proteins, roller bottle cultures wereseeded at 2×10⁵ cells/ml and incubated in a roller bottle incubator at37° C. under 5% CO₂ until the cell viability dropped below 25% (˜10days). Culture broth was clarified by centrifugation, filtered, andconcentrated up to 50-fold by ultrafiltration. For purification ofC_(H3)-AD2-IgG modules, concentrated supernatant fluid was loaded onto aProtein-A (MAB Select) affinity column. The column was washed tobaseline with PBS and the fusion proteins were eluted with 0.1 MGlycine, pH 2.5.

Example 3 Generation of DDD-Module Based on Interferon (IFN)-α2b

Construction of IFN-α2b-DDD2-pdHL2 for Expression in Mammalian Cells

The cDNA sequence for IFN-α2b was amplified by PCR resulting insequences comprising the following features, in which XbaI and BamHI arerestriction sites, the signal peptide is native to IFN-α2b, and 6 His isa hexahistidine tag: XbaI—Signal peptide—IFNα2b—6 His—BamHI (6 Hisdisclosed as SEQ ID NO: 28). The resulting secreted protein consisted ofIFN-α2b fused at its C-terminus to a polypeptide of the followingsequence:

(SEQ ID NO: 23) KSHHHHHHGSGGGGSGGGCGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA.

PCR amplification was accomplished using a full length human IFNα2b cDNAclone (Invitrogen Ultimate ORF human clone cat#HORF01Clone ID IOH35221)as a template and the following oligonucleotides as primers:

IFNA2 Xba I Left (SEQ ID NO: 24)TCTAGACACAGGACCTCATCATGGCCTTGACCTTTGCTTTACTGG IFNA2 BamHI right(SEQ ID NO: 25) GGATCCATGATGGTGATGATGGTGTGACTTTTCCTTACTTCTTAAACTT TCTTGC

The PCR amplimer was cloned into the pGemT vector. A DDD2-pdHL2mammalian expression vector was prepared for ligation with IFN-α2b asfollows. The C_(H1)-DDD2-Fab-hMN-14-pdHL2 (Rossi et al., Proc Natl AcadSci USA 2006, 103:6841-6) vector was digested with Xba I and Bam HI,which removes all of the Fab gene sequences but leaves the DDD2 codingsequence. The IFN-α2b amplimer was excised from pGemT with Xba I and BamHI and ligated into the DDD2-pdHL2 vector to generate the expressionvector IFN-α2b-DDD2-pdHL2.

Mammalian Cell Expression of IFN-α2b-DDD2

IFN-α2b-DDD2-pdHL2 was linearized by digestion with Sal I and stablytransfected by electroporation into Sp/ESF myeloma cells (see U.S.patent application Ser. No. 11/877,728, the Examples section of which isincorporated herein by reference). Two clones were found to havedetectable levels of IFN-α2b by ELISA. One of the two clones, designated95, was adapted to growth in serum-free media without substantialdecrease in productivity. The clone was subsequently amplified withincreasing MTX concentrations from 0.1 to 0.8 μM over five weeks. Atthis stage, it was sub-cloned by limiting dilution and the highestproducing sub-clone (95-5) was expanded. The productivity of 95-5 grownin shake-flasks was estimated to be 2.5 mg/L using commercial rIFN-α2b(Chemicon IF007, Lot 06008039084) as standards.

Purification of IFN-α2b-DDD2 from Batch Cultures Grown in Roller Bottles

Clone 95-5 was expanded to 34 roller bottles containing a total of 20 Lof serum-free Hybridoma SFM with 0.8 μM MTX and allowed to reachterminal culture. The culture broth was processed and IFN-α2b-DDD2 waspurified by immobilized metal affinity chromatography (IMAC) as follows.The supernatant fluid was clarified by centrifugation, 0.2 μM filtered,diafiltered into 1× Binding buffer (10 mM imidazole, 0.5 M NaCl, 50 mMNaH₂PO₄, pH 7.5), concentrated to 310 mL, added Tween 20 to a finalconcentration of 0.1%, and loaded onto a 30-mL Ni-NTA column. Followingsample loading, the column was washed with 500 mL of 0.02% Tween 20 in1× binding buffer and then 290 mL of 30 mM imidazole, 0.02% Tween 20,0.5 M NaCl, 50 mM NaH₂PO₄, pH 7.5. The product was eluted with 110 mL of250 mM imidazole, 0.02% Tween 20, 150 mM NaCl, 50 mM NaH₂PO₄, pH 7.5.Approximately 6 mg of IFNα2b-DDD2 was purified.

Production of IFN-α2b-DDD2 in E. coli

IFN-α2b-DDD2 was also expressed by microbial fermentation as a solubleprotein in E. coli. The coding sequence was amplified by PCR usingIFN-α2b-DDD2-pdHL2 DNA as a template. The amplimer was cloned into thepET26b E. coli expression vector using Nde I and Xho I restrictionsites. Protein was expressed intracellularly in BL21pLysS host cells byinduction of LB shake flasks with 100 μM IPTG at 18° C. for 12 hours.Soluble IFN-α2b-DDD2 was purified from cell lysates by IMAC as describedabove.

Example 4 Generation of a DNL Conjugate Comprising Four IFN-α2b-DDD2Moieties Linked to C_(H3)-AD2-IgG

A DNL complex comprising four IFN-α2b-DDD2 moieties linked toC_(H3)-AD2-IgG (FIG. 1) was made as follows. Briefly, a selectC_(H3)-AD2-IgG was combined with approximately two mole-equivalents ofIFN-α2b-DDD2 and the mixture was reduced under mild conditions overnightat room temperature after adding 1 mM EDTA and 2 mM reduced glutathione(GSH). Oxidized glutathione was added to 2 mM and the mixture was heldat room temperature for an additional 12-24 hours. The DNL conjugate waspurified over a Protein A affinity column. Four such DNL conjugatesdesigned 20-2b, 22-2b, hR1-2b, and 243-2b, each comprising four copiesof IFN-α2b anchored on C_(H3)-AD2-IgG-hA20 (with specificity for CD20),C_(H3)-AD2-IgG-hLL2 (with specificity for CD22), C_(H3)-AD2-IgG-hR1(with specificity for IGF-1R) and C_(H3)-AD2-IgG-hL243 (with specificityfor HLA-DR), respectively, were prepared. SE-HPLC analyses of 20-2bgenerated from mammalian (m) or E. coli (e)-produced IFN-α2b-DDD2 eachshowed a major peak having a retention time consistent with a covalentcomplex composed of an IgG and 4 IFN-α2b groups (not shown). SimilarSE-HPLC profiles were observed for the other three IFN-IgG conjugates.

Example 5 In Vitro Activity of the IFN-IgG Conjugates

The in vitro IFNα biological activity of 20-2b was compared to that ofcommercial PEGylated IFNα2 agents, PEGASYS® and PEG-INTRON®, usingcell-based reporter, viral protection, and lymphoma proliferationassays. Specific activities were determined using a cell-based kit,which utilizes a transgenic human pro-monocyte cell line carrying areporter gene fused to an interferon-stimulated response element (FIG.2A-2D). The specific activity of 20-2b (5300 IU/pmol) was greater thanboth PEGASYS® (170 IU/pmol) and PEG-INTRON® (3400 IU/pmol) (FIG. 2A).734-2b,1R-2b and five additional MAb-IFNα constructs (data not shown),which were produced similarly to 20-2b, each exhibited similar specificactivities (4000-8000 IU/pmol), demonstrating the consistency of the DNLmethod for generating such structures (FIG. 2A). Having four IFNα2bgroups contributed to the enhanced potency of MAb-IFNα. When normalizedto IFNα equivalents, the specific activity/IFNα was about 10-foldgreater than PEGASYS® and only about 2-fold less than PEG-INTRON®.

Comparison of MAb-IFNα, PEGASYS® and PEG-INTRON® in an in vitro viralprotection assay demonstrated that MAb-IFNα retains IFNα2b antiviralactivity with specific activities similar to PEG-INTRON® and 10-foldgreater than PEGASYS® (FIG. 2B).

IFNα2b can have a direct antiproliferative or cytotoxic effect on sometumor lines. The activity of 20-2b was measured in an in vitroproliferation assay with a Burkitt lymphoma cell line (Daudi) that ishighly sensitive to IFNα (FIG. 2C). Each of the IFNα2 agents efficientlyinhibited (>90%) Daudi in vitro with high potency (EC₅₀=4-10 pM).However, 20-2b (EC₅₀=0.25 pM) was about 30-fold more potent than thenon-targeting MAb-IFNα constructs. The parent anti-CD20 MAb of 20-2b hasanti-proliferative activity in vitro on many lymphoma cell lines,including Daudi (Rossi et al., 2008, Cancer Res 68:8384-92), atconsiderably greater concentrations (EC₅₀>10 nM). The in vitro activityof 20-2b was also assessed using Jeko-1, which is a mantle cell lymphomaline that has lower sensitivity to both IFNα and anti-CD20 (FIG. 2D).Jeko-1 is only modestly sensitive to the parent anti-CD20 MAb, having10% maximal inhibition (I_(max)) with an EC₅₀ near 1 nM. As shown with734-2b, Jeko-1 (I_(max)=43%; EC₅₀=23 pM) is less responsive to IFNα2bthan Daudi (I_(max)=90%; EC₅₀=7.5 pM). Compared to 734-2b, 20-2binhibited Jeko-1 to a greater extent (I_(max)=65%) and exhibited abiphasic dose-response curve (FIG. 2D). At <10 pM, a low-concentrationresponse attributed to IFNα2b activity was observed, which plateaus atI_(max)=43%, similar to 734-2b. A high-concentration response wasevident above 100 pM, where I_(max) reached 65%. The low-concentrationIFNα2b response of 20-2b (EC₅₀=0.97 pM) was 25-fold more potent than734-2b, similar to the results with Daudi.

A combination of the parent anti-CD20 antibody and 734-2b (v-mab+734-2b)was assayed to elucidate whether the increased potency of 20-2b is dueto an additive/synergistic effect of CD20 and IFNα signaling. The doseresponse curve for v-mab+734-2b was largely similar to 734-2b alone,except at >1 nM, where inhibition increased for the former but not thelatter. These results suggest that MAb targeting is responsible for thelower EC₅₀ of 20-2b, but its greater is apparently due to the additiveactivity of IFNα2b and CD-20 signaling. The effect of CD20 signaling wasonly evident in the high-concentration response for 20-2b (EC₅₀=0.85nM), which parallels the response to v-mab (EC₅₀=1.5 nM). A biphasicdose-response curve was not obvious for v-mab+734-2b, because the tworesponses overlap. However, an additive effect was evident at >1 nMconcentrations. The I_(max) of 20-2b (65%) was greater than the addedresponses of IFNα2b (I_(max)=43%) and v-mab (I_(max)=10%), suggestingpossible synergism between the actions of IFNα2b and v-mab.

ADCC Activity

IFNα can potentiate ADCC activity, which is a fundamental mechanism ofaction (MOA) for anti-CD20 immunotherapy, by activating NK cells andmacrophages. We compared ADCC of 20-2b and v-mab with two NHL cell linesusing peripheral blood mononuclear cells (PBMCs) as effector cells.Replicate assays using PBMCs from multiple donors consistentlydemonstrated that 20-2b had enhanced ADCC compared to v-mab, as shownfor both Daudi and Raji cells (FIG. 4A). This effect was also shown with22-2b, a MAb-IFNα comprising the anti-CD22 MAb, epratuzumab, which showsmodest ADCC (Carnahan et al., 2007, Mol Immunol 44:1331-41.

CDC Activity

CDC is thought to be an important MOA for Type-I anti-CD20 MAbs(including v-mab and rituximab). However, this function is lacking inthe Type-II MAbs, represented by tositumomab (Cardarelli et al., 2002,Cancer Immunol Immunother 51:15-24), which nonetheless has anti-lymphomaactivity. Unlike v-mab, 20-2b does not show CDC activity in vitro (FIG.4B). These results are consistent with those for other DNL structuresbased on the C_(H3)-AD2-IgG-v-mab module, in which complement fixationis apparently impaired, perhaps by steric interference (Rossi et al.,2008).

Example 6 Pharmacokinetic (PK) Analysis of 20-2b

The pharmacokinetic (PK) properties of 20-2b were evaluated in maleSwiss-Webster mice and compared to those of PEGASYS®, PEG-INTRON andα2b-413 (Pegylated IFN made by DNL, see U.S. patent application Ser. No.11/925,408). Concentrations of IFN-α in the serum samples at varioustimes were determined by ELISA following the manufacturer'sinstructions. Briefly, the serum samples were diluted appropriatelyaccording to the human IFN-α standard provided in the kit. An antibodybound to the microtiter plate wells captures interferon. A secondantibody was then used to reveal the bound interferon, which wasquantified by anti-secondary antibody conjugated to horseradishperoxidase (HRP) following the addition of Tetramethyl benzidine (TMB).The plates were read at 450 nm. FIG. 3 presents the results of the PKanalysis, which showed significantly slower elimination and longer serumresidence of 20-2b compared to the other agents. At an injected dose of210 pmol, the calculated pharmacokinetic serum half-life in hours was8.0 hr (20-2b), 5.7 hr (α2b-413), 4.7 hr (PEGASYS®) and 2.6 hr(PEG-INTRON). The elimination rate (1/h) was 0.087 (20-2b), 0.121(α2b-413), 0.149 (PEGASYS®) and 0.265 (PEG-INTRON). The calculatedMRT_(0.08)→∞ (hr) was 22.2 (20-2b), 12.5 (α2b-413), 10.7 (PEGASYS®) and6.0 (PEG-INTRON). Because the pharmacokinetic parameters are determinedmore by the nature of the complex than the individual antibody orcytokine, it is expected that the PK characteristics of the cytokine-DNLcomplex are generalizable to other cytokine moieties and antibodymoieties and are not limited to the specific 20-2b construct discussedabove.

Example 7 In Vivo Activity of 20-2b

Serum Stability.

20-2b was stable in human sera (>10 days) or whole blood (>6 days) at37° C. (not shown). Concentration of 20-2b complex was determined usinga bispecific ELISA assay. There was essentially no detectable change inserum 20-2b levels in either whole blood or serum over the time periodof the assay.

Ex Vivo Efficacy of 20-2b Against Lymphoma Cells from Whole Human Blood

We compared the abilities of 20-2b, v-mab, 734-2b, or v-mab+734-2b toeliminate lymphoma or normal B-cells from whole blood in an ex vivosetting (FIG. 5). The therapeutic efficacy of naked anti-CD20 MAbs isbelieved to be achieved via three mechanisms of action(MOA)—signaling-induced apoptosis or growth arrest, ADCC, and CDC(Glennie et al., 2007, Mol Immunol 44:3823-37). In this assay, v-mab canemploy all three MOA, while, based on the in vitro findings, 20-2b canpotentially take advantage of signaling and enhanced ADCC, but not CDC.In this short-term model, the IFNα2b groups of 20-2b and 734-2b can actdirectly on tumor cells, augment the ADCC activity of v-mab, andpossibly have some immunostimulatory effects. However, the full spectrumof IFNα-mediated activation of the innate and adaptive immune systemsthat might occur in vivo is not realized in this two-day ex vivo assay.

At 0.01 nM, 20-2b depleted Daudi cells (60.5%) significantly more thanv-mab (22.8%), 734-2b (38.6%) or v-mab+734-2b (41.7%) (FIG. 5). At 0.1nM, 20-2b and v-mab+734-2b depleted Daudi to a similar extent (88.9%),which was more than for v-mab (82.4%) or 734-2b (40.7%) (FIG. 5). At 1nM, each agent depleted Daudi >95%, except for 734-2b (55.7%) (FIG. 5).Each of the differences indicated were statistically significant(P<0.01).

Ramos is less sensitive than Daudi to both IFNα2b and v-mab. The effectof 734-2b was only moderate, resulting in <20% depletion of Ramos ateach concentration (FIG. 5). At both 0.01 and 0.1 nM, 20-2b depletedRamos more than v-mab+734-2b, which in turn eliminated more cells thanv-mab (FIG. 5). At 1 nM, all treatments besides 734-2b resulted insimilar Ramos depletion (75%) (FIG. 5). Each of the differencesindicated were statistically significant (P<0.02).

As demonstrated with 734-2b, IFNα2b alone does not deplete normalB-cells in this assay. At these low concentrations, 20-2b, v-mab, andv-mab+734-2b each show similar dose-responsive depletion of B-cells,which is markedly less than the depletion of either Daudi or Ramos. Noneof the treatments resulted in significant depletion of T-cells (data notshown).

In Vivo Efficacy of 20-2b in SCID Mice

A limitation of the mouse model is the very low sensitivity of murinecells to human IFNα2b. The overall therapeutic advantage of 20-2b thatmight be achieved in humans can involve the enhancement of both innateand adaptive immunity. With these limitations in mind, we studied theanti-lymphoma in vivo efficacy of 20-2b against disseminated Burkittlymphoma models in SCID mice. We initially tested a highly sensitiveearly Daudi model (FIG. 6A). One day after inoculation, groups wereadministered a single low dose of 20-2b, v-mab, or 734-2b. A single doseof v-mab or 734-2b at 0.7 pmol (170 ng) resulted in significantimprovement in survival when compared to saline for v-mab (P<0.0001),but not for the irrelevant MAb-IFNα control, 734-2b (FIG. 6A). Thisimprovement was modest, with the median survival time (MST) increasingfrom 27 days for saline to 34 days for v-mab. However, a single dose of0.7 pmol (170 ng) of 20-2b improved the MST by more than 100 days overboth saline control and v-mab groups (P<0.0001) (FIG. 6A). The study wasterminated after 19 weeks, at which time the 7 long-term survivors (LTS)in the 0.7 pmol 20-2b treatment group were necropsied with no visibleevidence of disease found (cured) (FIG. 6A). Remarkably, even the lowestdose of 0.07 pmol (17 ng) of 20-2b more than doubled the MST (FIG. 6A).

Next, we assessed the efficacy of 20-2b in a more challenging advancedDaudi model, in which mice were allowed to develop a substantiallygreater tumor burden prior to treatment (FIG. 6B). Seven days aftertumor inoculation, groups were administered a single low dose (0.7, 7.0or 70 pmol) of 20-2b, v-mab, 734-2b, or PEGASYS®. The MST for the salinecontrol mice was 21 days (FIG. 6B). The highest dose (70 pmol) ofPEGASYS® or 734-2b, each of which have enhanced Pk (compared torecombinant IFNα2b) but do not target tumor, doubled the MST (42 days;P<0.0001) (FIG. 6B). Treatment with 20-2b at a 100-fold lower dose (0.7pmol) produced similar results (38.5 days) as the highest dose (70 pmol)of either PEGASYS® or 734-2b (FIG. 6B). Treatment with 20-2b at a10-fold lower dose (7 pmol) resulted in significantly improved survival(80.5 days, 20% LTS) over treatment with 70 pmol of PEGASYS® or 734-2b(P<0.0012) (FIG. 6B). At the highest dose tested (70 pmol), 20-2bimproved the MST to >105 days with 100% LTS (FIG. 6B). We havedemonstrated previously with the early tumor model that v-mab canincrease survival of Daudi-bearing mice at relatively low doses (3.5pmol) while higher doses result in LTS. However, in this advanced tumormodel, a single dose of 70 pmol of v-mab had only a modest, thoughsignificant, effect on survival (MST=24 days, P=0.0001) (FIG. 6B).

We subsequently assayed 20-2b in more challenging models, which are lesssensitive than Daudi to direct inhibition by IFNα and less responsive toimmunotherapy with v-mab. Raji is ˜1000-fold less sensitive to thedirect action of IFNα2b compared to Daudi. However, Raji has a similarCD20 antigen density to Daudi (Stein et al., 2006, Blood 108:2736-44)and is responsive to v-mab, albeit considerably less so than Daudi(Goldenberg et al., 2009, Blood 113, 1062-70). The efficacy of 20-2b wasstudied in an advanced Raji model with therapy beginning five days aftertumor inoculation (FIG. 7A). Groups were administered a total of 6injections (250 pmol each) over two weeks. 734-2b did not improvesurvival over saline (MST=16 days), consistent with the insensitivity ofRaji to IFNα (FIG. 7A). V-mab significantly improved survival oversaline (MST=26 days, P<0.0001) (FIG. 7A). 20-2b was superior to allother treatments (MST=33 days, P<0.0001) (FIG. 7A).

Finally, we investigated the efficacy of 20-2b with NAMALWA (FIG. 7B), ahuman lymphoma that has low sensitivity to the direct action of IFNα,˜25-fold lower CD20 antigen density compared to Daudi or Raji, and isconsidered to be resistant to anti-CD20 immunotherapy (Stein et al.,2006). Groups were administered a total of 6 doses (250 pmol each) ofeither 20-2b or 734-2b. Another group was administered a total of 7doses (3.5 nmol each) of v-mab. The group treated with saline had an MSTof 17 days (FIG. 7B). Treatment with 734-2b very modestly, thoughsignificantly, improved survival (MST=20 days, P=0.0012) (FIG. 7B).20-2b (MST=34 days) was superior to 734-2b (P=0.0004) as well as v-mab(MST=24 days, P=0.0026), which was given at a 14-fold higher dose (FIG.7B).

Conclusions

The results demonstrate unequivocally that targeting of IFNα with ananti-CD20 MAb makes the immunocytokine more potent and effective thaneither agent alone or in combination. MAb targeting of IFNα to tumorsmay allow a less frequent dosing schedule of a single agent, reduce oreliminate side effects associated with IFN therapy, and result inprofoundly enhanced efficacy. Additionally, targeted IFNα can induce anacute tumor-directed immune response and possibly evoke immune memoryvia pleiotropic stimulation of innate and adaptive immunity (Belardelliet al, 2002, Cytokine Growth Factor Rev 12:119-34). Other groups haveproduced MAb-IFNα made by chemical conjugation that revealed some of thepotential clinical benefits of such constructs (Pelham et al., 1983,Cancer Immunol Immunother 15:210-16; Ozzello et al., 1998, Breast CancerRes Treat 48:135-47). A recombinant MAb-IFNα comprising murine IFNα andan anti-HER2/neu MAb exhibited potent inhibition of a transgenic(HER2/neu) murine B-cell lymphoma in immunocompetent mice and was alsocapable of inducing a protective adaptive immune response withimmunologic memory (Huang et al., 2007, J Immunol 179:6881-88).

We expect that therapy with 20-2b will stimulate localized recruitmentand activation of a number of immune cells, including NK, T4, T8, anddendritic cells, resulting in enhanced cytotoxicity and ADCC, and maypotentially induce tumor-directed immunologic memory. However, murinecells are exceedingly less sensitive (−4 logs) than human cells to humanIFNα2b (Kramer et al., 1983, J Interferon Res 3:425-35; Weck et al.,1981, J Gen Virol 57:233-37). Therefore, very little, if any, of theanti-lymphoma activity of 20-2b in the mouse model in vivo studiesdescribed above can be attributed to IFNα2b activation of the mouseimmune response. Rather, killing is due primarily to the direct actionof IFNα2b on the lymphoma cells.

We have shown that 20-2b has augmented ADCC, which may be the mostimportant MOA of anti-CD20 immunotherapy. However, since human IFNα2b isonly a very weak stimulator of the murine host's immune effector cells,an IFNα-enhanced ADCC is probably not realized as it might be in humans.Even with these limitations, the in vivo results demonstrate that 20-2bcan be a highly effective anti-lymphoma agent, exhibiting more than100-times the potency of v-mab or a non-targeting MAb-IFNα in theIFNα-sensitive Daudi model. Even with lymphoma models that arerelatively insensitive to the direct action of IFNα (Raji/NAMALWA) orare resistant to anti-CD20 immunotherapy (NAMALWA), 20-2b showedsuperior efficacy to either v-mab or non-targeted MAb-IFNα.

Fusion of IFNα2b to v-mab increases its in vivo potency by extendingcirculation times and enabling tumor targeting. The therapeuticsignificance of Pk was demonstrated in the Daudi model, where the slowerclearing PEGASYS® was superior to the faster clearing PEG-INTRON®, whichhas a higher specific activity (data not shown). 20-2b was considerablymore potent than either PEGASYS® or 734-2b, suggesting that lymphomatargeting via the anti-CD20 MAb is critical to its superior potency andefficacy. Surprisingly, the impact of targeting was evident even in thein vitro assays. In the in vitro proliferation experiments, which onlyallow for lymphoma inhibition via signaling, 20-2b showed activity at a25-fold lower concentration compared to non-targeting MAb-IFNα, eitheralone or when combined with v-mab. The ex vivo setting allows theinvolvement of all three of the anti-CD20 MOA. Even without CDCactivity, 20-2b was more effective at depleting lymphoma from blood thanIFNα or v-mab, either alone or in combination, demonstrating thesignificance of targeting. The influence of MAb targeting in the invitro/ex vivo studies is somewhat surprising, because the MAbs,effector, and target cells are all confined throughout the experiments.We expect that 20-2b will have a substantially greater impact in vivo inhuman patients.

The IFNα2b and v-mab components of 20-2b can apparently act additivelyor synergistically, to contribute to its enhanced potency. The in vitroproliferation assays suggest at least an additive effect, which wassubstantiated with the results of the ex vivo studies where thecombination of v-mab and 734-2b was superior to either agent alone. Thismay be accomplished ex vivo via increased ADCC activity of v-mab as partof 20-2b or when combined with 734-2b, yet ADCC is not functional in thein vitro proliferation assays, suggesting additional mechanisms. Thesignal transduced by v-mab-bound CD20 may potentiate the IFNα signal,resulting in enhanced potency. Alternatively, the binding of v-mab,which is a slowly internalizing MAb, may prevent theinternalization/down-regulation of the Type-I IFN receptors, resultingin a more prolonged and effective IFNα-induced signal.

Example 8 Generation of DDD Module Based on Erythropoietin (EPO)

Construction of EPO-DDD2-pdHL2 for Expression in Mammalian Cells

The cDNA sequence for EPO was amplified by PCR resulting in sequencescomprising the following features, in which Xba I and Bam HI arerestriction sites, the signal peptide is native to human EPO, and 6 Hisis a hexahistidine tag: XbaI—Signal peptide—EPO—6 His—-BamHI (6 Hisdisclosed as SEQ ID NO: 28). The resulting secreted protein consists ofEPO fused at its C-terminus to a polypeptide consisting of:

(SEQ ID NO: 23) KSHHHHHHGSGGGGSGGGCGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA

PCR amplification was accomplished using a full-length human EPO cDNAclone as a template and the following oligonucleotides as primers:

EPO Xba I left (SEQ ID NO: 26) TCTAGACACAGGACCTCATCATGGGGGTGCACGAATGTCCEPO BamHI Right (SEQ ID NO: 27)GGATCCATGATGGTGATGATGGTGTGACTTTCTGTCCCCTGTCCTGCAG

The PCR amplimer was cloned into the pGemT vector. A DDD2-pdHL2mammalian expression vector was prepared for ligation with EPO bydigestion with XbaI and Bam HI restriction endonucleases. The EPOamplimer was excised from pGemT with XbaI and Bam HI and ligated intothe DDD2-pdHL2 vector to generate the expression vector EPO-DDD2-pdHL2.

Mammalian Cell Expression of EPO-DDD2

EPO-pdHL2 was linearized by digestion with SalI enzyme and stablytransfected into Sp/ESF myeloma cells by electroporation. Clones wereselected with media containing 0.15 μM MTX. Clones #41, 49 and 37 eachwere shown to produce ˜0.5 mg/L of EPO by an ELISA using NuncImmobilizer Nickel-Chelate plates to capture the His-tagged fusionprotein and detection with anti-EPO antibody. Approximately 2.5 mg ofEPO-DDD2 was purified by IMAC from 9.6 liters of serum-free rollerbottle culture.

Example 9 Generation of 734-EPO, a DNL Conjugate Comprising FourEPO-DDD2 Moieties Linked to C_(H3)-AD2-IgG-h734

734-EPO was produced as described above for 20-2b. SE-HPLC analysis ofthe protein A-purified 734-EPO showed a major peak and a shoulder of ahigher molecular size (not shown). The retention time of the major peakwas consistent with a covalent complex composed of an IgG and 4 EPOgroups. The shoulder was likely due to a non-covalent dimer of theIgG-EPO conjugate. SDS-PAGE analysis with Coomassie blue staining andanti-EPO immunoblot analysis showed that under non-reducing conditionsthe product had a Mr of >260 kDa (not shown), consistent with thededuced MW of ˜310 kDa. Under reducing conditions the bands representingthe three constituent polypeptides of 734-EPO (EPO-DDD2, Heavychain-AD2, and light chain) were evident and appeared to be similar inquantity (not shown). Non-product contaminants were not detected.

EPO-DDD2 and 734-EPO were assayed for their ability to stimulate thegrowth of EPO-responsive TF1 cells (ATCC) using recombinant human EPO(Calbiochem) as a positive control. TF1 cells were grown in RPMI 1640media supplemented with 20% FBS without GM-CSF supplementation in96-well plates containing 1×10⁴ cells/well. The concentrations(units/ml) of the EPO constructs were determined using a commercial kit(Human erythropoietin ELISA kit, Stem Cell Research, Cat#01630). Cellswere cultured in the presence of rhEPO, EPO-DDD2 or 734-EPO atconcentrations ranging from 900 u/ml to 0.001 U/ml for 72 hours. Theviable cell densities were compared by MTS assay using 20 μl of MTSreagent/well incubated for 6 hours before measuring the OD490 in a96-well plate reader. Dose response curves and EC₅₀ values weregenerated using Graph Pad Prism software (FIG. 8). Both EPO-DDD2 and734-EPO showed in vitro biological activity that was approximately 10%of rhEPO.

Example 10 Generation of DDD Module Based on Granulocyte-ColonyStimulating Factor (G-CSF)

Construction of G-CSF-DDD2-pdHL2 for Expression in Mammalian Cells

The cDNA sequence for G-CSF was amplified by PCR, resulting in sequencescomprising the following features in which the signal peptide is nativeto human G-CSF and 6 His is a hexahistidine tag: Xba I—Signalpeptide—G-CSF—6 His—Bam HI (6 His disclosed as SEQ ID NO: 28). Theresulting secreted protein consisted of G-CSF fused at its C-terminus toa polypeptide consisting of SEQ ID NO:3.

PCR amplification was accomplished using a full-length human G-CSF cDNAclone (Invitrogen IMAGE human cat#97002RG Clone ID 5759022) as atemplate and the following oligonucleotides as primers:

G-CSF XbaI Left (SEQ ID NO: 29)TCTAGACACAGGACCTCATCATGGCTGGACCTGCCACCCAG G-CSF BamHI-Right(SEQ ID NO: 30) GGATCCATGATGGTGATGATGGTGTGACTTGGGCTGGGCAAGGTGGCGT AG. 

The PCR amplimer was cloned into the pGemT vector. A DDD2-pdHL2mammalian expression vector was prepared for ligation with G-CSF bydigestion with XbaI and Bam HI restriction endonucleases. The G-CSFamplimer was excised from pGemT with XbaI and Bam HI and ligated intothe DDD2-pdHL2 vector to generate the expression vectorG-CSF-DDD2-pdHL2.

Mammalian Cell Expression of G-CSF-DDD2

G-CSF-pdHL2 was linearized by digestion with Sal I enzyme and stablytransfected into Sp/ESF myeloma cells by electroporation. Clones wereselected with media containing 0.15 μM MTX. Clone #4 was shown toproduce 0.15 mg/L of G-CSF-DDD2 by sandwich ELISA.

Purification of G-CSF-DDD2 from Batch Cultures Grown in Roller Bottles

Clone #4 was expanded to 34 roller bottles containing a total of 20 L ofHybridoma SFM with 0.4 μM MTX and allowed to reach terminal culture. Thesupernatant fluid was clarified by centrifugation, filtered (0.2 μM),diafiltered into 1× Binding buffer (10 mM Imidazole, 0.5 M NaCl, 50 mMNaH₂PO₄, pH 7.5 and concentrated. The concentrate was purified by IMAC.

Construction and Expression of G-CSF-DDD2 in E. coli.

G-CSF-DDD2 was also expressed by microbial fermentation as a solubleprotein in E. coli. The coding sequence was amplified by PCR usingG-CSF-DDD2-pdHL2 DNA as a template. The amplimer was cloned into thepET26b E. coli expression vector using Nde I and Xho I restrictionsites. Protein was expressed intracellularly in BL21pLysS host cells byinduction of LB shake flasks with 100 pM IPTG at 18° C. for 12 hours.Soluble G-CSF-DDD2 was purified from cell lysates by IMAC.

Construction and Expression of N-DDD2-G-CSF(C17S) in E. coli.

An alternative G-CSF-DDD2 module was made by fusing the DDD2 sequenceand a peptide spacer at the N-terminus of G-CSF(C17S), which differsfrom the wild-type by substituting the unpaired cysteine residue at the17^(th) position with a serine. N-DDD2-G-CSF(C17S) was expressed in E.coli and purified by IMAC.

Example 11 Generation of hR1-17S, a DNL Conjugate Comprising FourN-DDD2-G-CSF(C17S) Moieties Linked to C_(H3)-AD2-IgG-hR1

hR1-17S was produced by combining C_(H3)-AD2-IgG-hR1 with excessN-DDD2-G-CSF(C17S) under redox conditions following purification byProtein A affinity chromatography. SE-HPLC analysis of the proteinA-purified hR1-17S showed a major peak and a shoulder of a highermolecular size (not shown). The retention time of the major peak wasconsistent with a covalent complex composed of an IgG and 4 G-CSFgroups. The shoulder was likely due to a non-covalent dimer of theIgG-G-CSF conjugate. SDS-PAGE analysis with Coomassie blue staining andanti-G-CSF immunoblot analysis showed that under non-reducing conditionsthe product had an Mr consistent with the deduced MW of ˜260 kDa. Underreducing conditions, bands representing the three constituentpolypeptides of hR1-17S(N-DDD2-G-CSF(C17S), Heavy chain-AD2, and lightchain) were detected (not shown).

Example 12 Tetrameric G-CSF with Improved Biological Activity

The Dock-and-Lock (DNL) method was applied to produce three IgG-AD2fusion proteins, each of which was combined with the dimer of aG-CSF-DDD2 fusion protein to generate a stably tethered complexcomprising an intact IgG linked at its CH3 termini to four molecules ofG-CSF. The humanized IgGs fused to AD2 were hA20 (anti-CD20), hMN14(anti-CEACAM5), and h734 (anti-indium-DTPA). In G-CSF-DDD2, the unpairedcysteine (C17) of wild-type G-CSF was replaced with a serine. Asexpected, a dimeric form of G-CSF-DDD2 was predominantly obtainedfollowing purification. Reacting G-CSF-DDD2 to each of the three IgG-AD2modules resulted in hA20-G-CSF, hMN14-G-CSF and h734-G-CSF,respectively. IgG-G-CSF was purified from the reaction mixture byProtein A chromatography.

The purity, size, and composition of each IgG-G-CSF were confirmed bySDS-PAGE, size-exclusion HPLC, and western blotting (not shown). An invitro proliferation assay using Kasumi-1 (a myeloid leukemia cell line)showed that IgG-G-CSF complexes were more potent compared withrecombinant hG-CSF (FIG. 9). ADCC studies of hA20-G-CSF were performedin CD20-positive Daudi cells with the PBMCs isolated from two differentdonors (FIG. 10). For both donors, enhanced ADCC was observed forhA20-G-CSF compared to the parental hA20 IgG (veltuzumab): donor 006,35% vs. 20% (P<0.0191); donor 010, 60% vs. 50% (not statisticallysignificant). The concentration of each agent tested was 5 μg/mL.

In vivo studies in normal mice also revealed the spleen of the treatedgroup had an average weight that was about 2- to 3-times that of theuntreated group (P=0.0061) (FIG. 11). In addition, the numbers ofmonocytes and neutrophils in the blood of the treated mice, whenmeasured at day 9, were increased about 5- and 2-fold, respectively,compared to the untreated mice (FIG. 11). The three IgG-G-CSF complexesrepresent a novel class of bioactive immunocytokines comprisingtetrameric G-CSF anchored onto an IgG, which are expected to showimproved pharmacokinetics and additional targeting specificity conferredby the built-in IgG. Because rituximab (anti-CD20) therapy causesneutropenia in patients, the potential of hA20-IgG-G-CSF to enhance thepotency of an anti-CD20 antibody yet prevent neutropenia isadvantageous.

Example 13 Tetrameric Erythropoietin with Improved Biological Activity

We combined a recombinant fusion protein comprising erythropoietinattached to a DDD sequence with either (1) a recombinant 40 kDa PEG-ADmodule to generate a PEGylated dimeric Epo; (2) a recombinant Fab-ADmodule to generate a dimeric Epo-Fab construct; or (3) a recombinantIgG-AD module to generate a tetrameric Epo-IgG conjugate. Epo-DDD2 wasgenerated by recombinant fusion of the DDD2 peptide to the carbodylterminus of human Epo via an 18 amino acid linker peptide.C_(H)1-AD2-Fab was generated by recombinant fusion of the AD2 peptide tothe carboxyl terminus of the C_(H)1 domain of a Fab via a 15 amino acidlinker peptide. IMP457 was generated by forming a sulfide linkagebetween the maleimide group of mPEG2-MAL-40K to the N-terminal cysteineresidue of the AD2 peptide IMP 421.

IMP 421 (SEQ ID NO: 31)Ac-C-PEG₃-C(S-tBu)GQIEYLAKQIVDNAIQQAGC(S-tBu)G-NH₂

DNL modules for IgG-AD2, Fab-AD2 and Epo-DDD2 were purified from thesupernatant fluid of separate recombinant myeloma cultures by Protein A,Protein L and immobilized metal affinity chromatography (IMAC),respectively. Combining an IgG-AD2 module with slightly more than twomolar equivalents of Epo-DDD2 module under mild redox conditionsresulted in the formation of a covalent complex comprising one IgG and 4Epo groups via the docking of each of the two AD2 domains on IgG with adimer of Epo-DDD2, and subsequent formation of disulfide bonds (locking)between DDD2 and AD2. IgG-Epo, which was purified by Protein A followingthe DNL reaction, was detected by non-reducing SDS-PAGE andsize-exclusion HPLC (SE-HPLC), where it was resolved as a singlepredominant protein peak with a retention time consistent with a 255-kDaprotein (not shown). Combining Fab-AD2 and Epo-DDD2 modules by DNLresulted in the formation of a covalent complex comprising one Fab and 2Epo groups (not shown). Fab-Epo was purified by Protein L and analyzedby non-reducing SDS-PAGE and SE-HPLC, where it was resolved as a singlepredominant protein peak with a retention time consistent with a 100-kDaprotein (not shown). Combining a PEG-AD2 module (IMP457) and Epo-DDD2resulted in the formation of a covalent complex comprising 40 kDabranched PEG and 2 Epo groups. 457-Epo, which was purified byQ-Sepharose IEC at pH-6, was detected by non-reducing SDS-PAGE withCoomassie blue and Iodine staining (not shown).

The in vitro potency of various Epo constructs was measured by theirability to stimulate the proliferation of the cytokine-dependent TF1cell line (FIG. 12). TF1 cells were incubated with serial dilutions ofrhEpo, ARANESP®, Epo-DDD2, Fab-Epo, IgG-Epo, or 457-Epo. The results(FIG. 12) show that the Epo-DDD2 module and each DNL constructmaintained Epo biological activity. IgG-Epo, Fab-Epo and 457-Epo allstimulated of proliferation of the cytokine-dependent TF1 cell line withsimilar potency to rhEpo (ARANESP®).

In vivo activity of IgG-Epo was determined by its effects on hematocritlevels. Normal Swiss-Webster mice (n=4) were administered a single i.v.injection (3.2 μg) of IgG-Epo and the hematocrit was measured after fivedays. The results (FIG. 13) showed that a single i.v. administration ofIgG-hEpo produced a significant (P=0.0107) increase in hematocritcompared to untreated mice. The DNL method provides a novel approach forefficiently tethering cytokines, such as Epo, to a variety of moleculesincluding IgG, Fab and PEG, which may enhance their in vivo biologicalactivity by improving their pharmakokinetics and stability.

All of the COMPOSITIONS and METHODS disclosed and claimed herein can bemade and used without undue experimentation in light of the presentdisclosure. While the compositions and methods have been described interms of preferred embodiments, it is apparent to those of skill in theart that variations maybe applied to the COMPOSITIONS and METHODS and inthe steps or in the sequence of steps of the METHODS described hereinwithout departing from the concept, spirit and scope of the invention.More specifically, certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

What is claimed is:
 1. A method of delivering a cytokine comprising: a)obtaining a cytokine-antibody DNL complex comprising; (i) a cytokinemoiety attached to a DDD (dimerization and docking domain) moiety,wherein said DDD moiety is from a dimerization and docking domain ofprotein kinase A and consists of the amino acid sequence selected fromthe group consisting of SEQ ID NO:1 and SEQ ID NO:2; and (ii) anantibody moiety attached to an AD (anchor domain) moiety, wherein the ADmoiety is from an anchoring domain of an AKAP (A-kinase anchoringprotein) and consists of the amino acid sequence selected from the groupconsisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15 and SEQ ID NO:16, wherein the DDD moieties form adimer that binds to the AD moiety to form the complex; and b)administering the complex to a human subject.
 2. The method of claim 1,wherein the antibody moiety is selected from the group consisting of anIgG antibody and an antigen-binding antibody fragment thereof.
 3. Themethod of claim 1, wherein the antibody moiety binds to an antigenselected from the group consisting of carbonic anhydrase IX, CCL19,CCL21, CSAp, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15,CD16, CD18, CD19, IGF-1R, CD20, CD21, CD22, CD23, CD25, CD29, CD30,CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD55,CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126,CD133, CD138, CD147, CD154, AFP, PSMA, CEACAM5, CEACAM-6, B7, ED-B offibronectin, Factor H, Flt-1, Flt-3, folate receptor, GROB, HMGB-1,hypoxia inducible factor (HIF), HM1.24, insulin-like growth factor-1(ILGF-1), IFN-.gamma., IFN-.alpha., IFN-.beta., IL-2, IL-4R, IL-6R,IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18,IL-25, IP-10, MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3,MUC4, MUC5, PAM4 antigen, NCA-95, NCA-90, Ia, EGP-1, EGP-2, HLA-DR,tenascin, Le(y), RANTES, TAC, Tn antigen, Thomson-Friedenreich antigens,tumor necrosis antigens, TNF-.alpha., TRAIL receptor (R1 and R2), VEGFR,EGFR, P1GF, complement factors C3, C3a, C3b, C5a and C5.
 4. The methodof claim 3, wherein the antibody moiety is selected from the groupconsisting of hR1 (anti-IGF-1R), hPAM4 (anti-mucin), hA20 (anti-CD20),hA19 (anti-CD19), hIMMU31 (anti-AFP), hLL1 (anti-CD74), hLL2(anti-CD22), hMu-9 (anti-CSAp), hL243 (anti-HLA-DR), hMN-14 (anti-CEA),hMN-15 (anti-CEA), hRS7 (anti-EGP-1) and hMN-3 (anti-CEA).
 5. The methodof claim 1, wherein the complex comprises an IgG antibody and theC-terminal end of each heavy chain of the IgG antibody is attached to anAD moiety.
 6. The method of claim 5, wherein each AD moiety binds to twoDDD moieties and the complex comprises four cytokine moieties.
 7. Themethod of claim 1, wherein the cytokine moiety is selected from thegroup consisting of MIF (macrophage migration inhibitory factor), HMGB-1(high mobility group box protein 1), TNF-α, IL-1, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16,IL-17, IL-18, IL-19, IL-23, IL-24, CCL19, CCL21, MCP-1, RANTES, MIP-1A,MIP-1B, ENA-78, IP-10, Gro-β, Eotaxin, interferon-α, interferon-β,interferon-γ, G-CSF, GM-CSF, SCF, PDGF, MSF, Flt-3 ligand,erythropoietin, thrombopoietin, CNTF, leptin, oncostatin M, VEGF, EGF,FGF, P1GF, insulin, hGH, calcitonin, Factor VIII, IGF, somatostatin,tissue plasminogen activator and LIF.
 8. The method of claim 1, whereinthe cytokine moiety is selected from the group consisting of interferon(IFN)-α2b, G-CSF and erythropoietin.
 9. The method of claim 1, whereinthe antibody moiety is hA20 IgG antibody and the cytokine moiety ishuman IFNα2b.
 10. The method of claim 2, wherein a heavy chain of theIgG antibody and the AD moiety form a first fusion protein and thecytokine moiety and the DDD moiety form a second fusion protein.
 11. Thecomplex of claim 1, wherein the cytokine-antibody DNL complex exhibits alonger serum half-life than the cytokine alone and a PEGylated form ofthe cytokine.
 12. The method of claim 1, wherein the cytokine-antibodyDNL complex exhibits a greater efficacy than the cytokine moiety alone,the antibody moiety alone, the combination of unconjugated cytokinemoiety with unconjugated antibody moiety or a PEGylated form of thecytokine moiety.